Combinations comprising positive allosteric modulators or orthosteric agonists of metabotropic glutamatergic receptor subtype 2 and their use

ABSTRACT

The present invention relates to combinations comprising a positive allosteric modulator (“PAM”) of metabotropic glutamatergic receptor subtype 2 (“mGluR2”) or a pharmaceutically acceptable salt or a solvate thereof, or an orthosteric agonist of metabotropic glutamatergic receptor subtype 2 compound or a pharmaceutically acceptable salt or a solvate thereof, and a synaptic vesicle protein 2A (“SV2A”) ligand.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the national stage of PCT Application No.PCT/EP2015/051029, filed Jan. 20, 2015, which claims priority from U.S.Provisional Application No. 61/929,795, filed Jan. 21, 2014, EuropeanPatent Application No. 14153887.6, filed Feb. 4, 2014, European PatentApplication No. 14153880.1, filed Feb. 4, 2014, European PatentApplication No. 14183324.4, filed Sep. 3, 2014, European PatentApplication No. 14187429.7, filed Oct. 2, 2014 and U.S. ProvisionalPatent Application No. 62/091,668, filed Dec. 15, 2014, the entiredisclosures of which are hereby incorporated in their entirety.

FIELD OF THE INVENTION

The present invention relates to combinations comprising a positiveallosteric modulator (“PAM”) of metabotropic glutamatergic receptorsubtype 2 (“mGluR2”) or a pharmaceutically acceptable salt or a solvatethereof, or an orthosteric agonist of metabotropic glutamatergicreceptor subtype 2 compound or a pharmaceutically acceptable salt or asolvate thereof, and a synaptic vesicle protein 2A (“SV2A”) ligand.

BACKGROUND OF THE INVENTION

Epilepsy describes a condition in which a person has recurrent seizuresdue to a chronic, underlying process. Epilepsy refers to a clinicalphenomenon rather than a single disease entity, since there are manyforms and causes of epilepsy. Using a definition of epilepsy as two ormore unprovoked seizures, the incidence of epilepsy is estimated atapproximately 0.3 to 0.5 percent in different populations throughout theworld, with the prevalence of epilepsy estimated at 5 to 10 people per1000.

An essential step in the evaluation and management of a patient with aseizure is to determine the type of seizure that has occurred. The maincharacteristic that distinguishes the different categories of seizuresis whether the seizure activity is partial (synonymous with focal) orgeneralized.

Partial seizures are those in which the seizure activity is restrictedto discrete areas of the cerebral cortex. If consciousness is fullypreserved during the seizure, the clinical manifestations are consideredrelatively simple and the seizure is termed a simple-partial seizure. Ifconsciousness is impaired, the seizure is termed a complex-partialseizure. An important additional subgroup comprises those seizures thatbegin as partial seizures and then spread diffusely throughout thecortex, which are known as partial seizures with secondarygeneralization.

Generalized seizures involve diffuse regions of the brain simultaneouslyin a bilaterally symmetric fashion. Absence or petit mal seizures arecharacterized by sudden, brief lapses of consciousness without loss ofpostural control. Atypical absence seizures typically include a longerduration in the lapse of consciousness, less abrupt onset and cessation,and more obvious motor signs that may include focal or lateralizingfeatures. Generalized tonic-clonic or grand mal seizures, the main typeof generalized seizures, are characterized by abrupt onset, withoutwarning. The initial phase of the seizure is usually tonic contractionof muscles, impaired respiration, a marked enhancement of sympathetictone leading to increased heart rate, blood pressure, and pupillarysize. After 10-20 s, the tonic phase of the seizure typically evolvesinto the clonic phase, produced by the superimposition of periods ofmuscle relaxation on the tonic muscle contraction. The periods ofrelaxation progressively increase until the end of the ictal phase,which usually lasts no more than 1 min. The postictal phase ischaracterized by unresponsiveness, muscular flaccidity, and excessivesalivation that can cause stridorous breathing and partial airwayobstruction. Atonic seizures are characterized by sudden loss ofpostural muscle tone lasting 1-2 s. Consciousness is briefly impaired,but there is usually no postictal confusion. Myoclonic seizures arecharacterized by a sudden and brief muscle contraction that may involveone part of the body or the entire body.

The synaptic vesicle protein 2A (“SV2A”) has been identified as a broadspectrum anticonvulsant target in models of partial and generalizedepilepsy. Studies performed in animal models and human tissue suggestthat changes in the expression of SV2A are implicated in epilepsy (for areview see for instance: (a) Mendoza-Torreblanca et al. “Synapticvesicle protein 2A: basic facts and role in synaptic function” EuropeanJournal of Neuroscience 2013, pp. 1-11; (b) Kaminski R M, et al.“Targeting SV2A for Discovery of Antiepileptic Drugs”. In: Noebels J L,Avoli M, Rogawski M A, et al., editors. Jasper's Basic Mechanisms of theEpilepsies [Internet]. 4th edition. Bethesda (Md.): National Center forBiotechnology Information (US); 2012. Available from:http://www.ncbi.nlm.nih.gov/books/NBK98183/).

The exact role of SV2A remains unclear but studies suggest that changesin the expression of SV2A affect synaptic function (Nowack et al.“Levetiracetam reverses synaptic deficits produced by overexpression ofSV2A” PLoS One 2011, Volume 6 (12), e29560). It has also been suggestedthat SV2A is a key player in exocytosis and is involved inneurotransmission (Crowder et al. “Abnormal neurotransmission in micelacking synaptic vesicle protein 2A (SV2A)” Proc Nat Acad Sci USA 1999,96, pp. 15268-15273) and studies in knock-out mice suggest that lack ofSV2A results in an imbalance between glutamatergic and GABAergicneurotransmission (Venkatesan et al. “Altered balance between excitatoryand inhibitory inputs onto CA pyramidal neurons from SV2A-deficient butnot SV2B-deficient mice” J Neurosci Res 2012, 90, pp. 2317-2327).Decreased expression of SV2A may be a consequence of seizure activityand may be involved in the progression of epilepsy (van Vliet et al.“Decreased expression of synaptic vesicle protein 2A, the binding sitefor levetiracetam, during epileptogenesis and chronic epilepsy”Epilepsia 2009, 50, pp. 422-433; Feng et al. “Down-regulation ofsynaptic vesicle protein 2A in the anterior temporal neocortex ofpatients with intractable epilepsy” J Mol Neurosci 2009, 39, pp.354-359; Toering et al. “Expression patterns of synaptic vesicle protein2A in focal cortical dysplasia and TSC-cortical tubers” Epilepsia 2009,50, pp. 1409-1418) and epileptogenesis in patients with brain tumours(de Groot et al. “Expression of synaptic vesicle protein 2A inepilepsy-associated brain tumors and in the peritumoral cortex”Neuro-Oncology 2010, 12, pp. 265-273).

SV2A ligands include levetiracetam (Lynch et al. “The synaptic vesicleprotein SV2A is the binding site for the antiepileptic druglevetiracetam” Proc. Natl. Acad. Sci. USA 2004, Vol. 101, pp.9861-9866), brivaracetam and seletracetam (Kaminski R M, et al.“Targeting SV2A for Discovery of Antiepileptic Drugs”. In: Noebels J L,Avoli M, Rogawski M A, et al., editors. Jasper's Basic Mechanisms of theEpilepsies [Internet]. 4th edition. Bethesda (Md.): National Center forBiotechnology Information (US); 2012. Available from:http://www.ncbi.nlm.nih.gov/books/NBK98183/; Nowack et al.“Levetiracetam reverses synaptic deficits produced by overexpression ofSV2A” PLoSone December 2011, Vol. 6(12), e29560).

Levetiracetam, (−)-(S)-α-ethyl-2-oxo-1-pyrrolidine acetamide or(S)-2-(2-oxopyrrolidin-1-yl)butanamide,

is an antiepileptic drug. It showed no activity in traditional acutemodels (maximal electroshock and pentylenetetrazol seizure tests) butwas found potent in chronic epilepsy models and in genetic models ofgeneralized epilepsy. It has shown a high safety margin compared toother antiepileptic drugs (Klitgaard “Levetiracetam: the preclinicalprofile of a new class of antiepileptic drugs” Epilepsia 2001, 42(Supplement 4), pp. 13-18). It is commercialized under the trademarkKeppra®, available as tablets, as an oral solution, and as a concentratemade up into a solution for infusion. Keppra® has been approved inEurope as a monotherapy in patients from 16 years of age with newlydiagnosed epilepsy, in the treatment of partial-onset seizures (fits)with or without secondary generalization and as an add-on therapy foruse with other anti-epileptic drugs in the treatment of partial-onsetseizures with or without generalization in patients from 1 month of age;myoclonic seizures in patients from 12 years of age with juvenilemyoclonic epilepsy; and primary generalized tonic-clonic seizures inpatients from 12 years of age with idiopathic generalized epilepsy(www.ema.europa.eu). Keppra® has also been approved in the USA as anadd-on therapy for the treatment of partial onset seizures in patientsfrom 1 month of age; myoclonic seizures in patients 12 years of age andolder with juvenile myoclonic epilepsy; and primary generalizedtonic-clonic seizures in patients 6 years of age and older withidiopathic generalized epilepsy. Keppra XR®, available asextended-release tablets, has been approved in the USA for the add-ontreatment of partial onset seizures in patients 16 years of age andolder with epilepsy(http://www.accessdata.fda.gov/scripts/cder/drugsatfda/index.cfm).

Brivaracetam, the 4-n-propyl analog of levetiracetam,(2S)-2-[(4R)-oxo-4-propyl-pyrrolidin-1-yl]butanamide,

is in clinical trials and investigated as monotherapy in partial onsetseizures and post-herpetic neuralgia and as add-on therapy in refractorypartial onset seizures, Unverricht-Lundborg disease in adolescents andadults and in photosensitive epilepsy (www.clinicaltrials.gov).

Seletracetam,(2S)-2-[(4S)-4-(2,2,-difluorovinyl)-2-oxo-pyrrolidin-1-yl]butanamide,

has been tested in clinical trials.

Processes for the preparation of the three compounds are known in theliterature. For instance, processes for making Levetiracetam aredisclosed for instance, in EP 0 162 036 and in GB 2 225 322. A processfor the preparation of Brivaracetam is disclosed for instance in WO01/62726. A process for the preparation of Seletracetam is known forinstance from WO2005/121082. Alternative processes for making the threecompounds are disclosed in EP1806339.

Antiepileptic drugs have found usefulness in neurological andpsychiatric disorders, including neuropathic pain, migraine, essentialtremor and in anxiety, schizophrenia and bipolar disorder (Landmarck“Antiepileptic drugs in non-epilepsy disorders. Relations betweenmechanisms of action and clinical efficacy” CNS Drugs 2008, Vol. 22(1),pp. 27-47; Calabresi et al. “Antiepileptic drugs in migraine: fromclinical aspects to cellular mechanisms” Trends in PharmacologicalSciences 2007, Vol. 28(4), pp. 188-195; Rogawski and Löscher “Theneurobiology of antiepileptic drugs for the treatment of nonepilepticconditions” Nat Med 2004, Vol. 10, pp. 685-692).

Levetiracetam has been found effective or potentially effective in awide-spectrum of neuropsychiatric disorders including mood disorders(Muralidharan and Bhagwagar “Potential of levetiracetam in mooddisorders: a preliminary review” CNS Drugs 2006, Vol. 20, pp. 969-979;Mula et al. “The role of anticonvulsant drugs in anxiety disorders: acritical review of the evidence” J Clin Pshycopharmacol 2007, Vol. 27,pp. 263-272), anxiety disorders (Kinrys et al. “Levetiracetam asadjunctive therapy for refractory anxiety disorders” J Clin Psychiatry2007, Vol. 68, pp. 1010-1013; Zhang et al. “Levetiracetam in socialphobia: a placebo controlled pilot study” J Psychopharmacol 2005, Vol.19, pp. 551-553; Kinrys et al. “Levetiracetam for treatment-refractoryposttraumatic stress disorder” J Clin Psychiatry 2006, Vol. 67, pp.211-214), pain (Enggaard et al. “Specific effect of levetiracetam inexperimental human pain models” Eur J Pain 2006, Vol. 10, pp. 193-198;Dunteman “Levetiracetam as an adjunctive analgesic in neoplasticplexopathies: case series and commentary” J Pain Palliative CarePharmacother 2005, Vol. 19, pp. 35-43; Price “Levetiracetam in thetreatment of neuropathic pain: three case studies” Clin J Pain 2004,Vol. 20, pp. 33-36), movement disorders (Bushara et al. “The effect oflevetiracetam on essential tremor” Neurology 2005, Vol. 64, pp.1078-1080; McGavin et al “Levetiracetam as a treatment for tardivedyskinesia: a case report” Neurology 2003, Vol. 61, pp. 419; Woods etal. “Effects of levetiracetam on tardive dyskinesia: a randomized,double-blind, placebo-controlled study” J Clin Psychiatry 2008, Vol. 69,pp. 546-554; Zivkovic et al. “Treatment of tardive dyskinesia withlevetiracetam in a transplant patient” Acta Neurol Scand 2008, Vol. 117,pp. 351-353; Striano et al. “Dramatic response to levetiracetam inpost-ischaemic Holmes' tremor” J Neurol Neurosurg Psychiatry 2007, Vol.78, pp. 438-439) and it is suspected to show potentially beneficialeffects in cognitive functioning (Piazzini et al. “Levetiracetam: Animprovement of attention and of oral fluency in patients with partialepilepsy” Epilepsy Research 2006, Vol. 68, pp. 181-188; de Groot et al.“Levetiracetam improves verbal memory in high-grade glioma patients”Neuro-oncology 2013, Vol. 15(2), pp. 216-223; Bakker et al. “Reductionof hippocampal hyperactivity improves cognition in amnestic mildcognitive impairment” Neuron 2012, Vol. 74, pp. 467-474; for a review:Eddy et al. “The cognitive impact of antiepileptic drugs” Ther AdvNeurol Disord 2011, Vol. 4(6), pp. 385-407 and references cited therein;Wheless “Levetiracetam in the treatment of childhood epilepsy”Neuropsychiatric Disease and Treatment 2007, Vol. 3(4), pp. 409-421),and behavioral symptoms in dementia (Dolder and Nealy “The efficacy andsafety of newer anticonvulsants in patients with dementia” Drugs Aging2012, Vol. 29(8), pp. 627-637). Animal data and some preliminaryclinical trials suggest that levetiracetam may have potential forrestraining post-traumatic epilepsy, such as those caused by statusepilepticus, traumatic brain injury and ischemic stroke, and it appearsto have neuroprotective effects. The potential of levetiracetam ineasing epileptogenesis or cognitive dysfunction remains to beascertained by conclusive animal and clinical studies (for reviews:Löscher and Brandt “Prevention or modification of epileptogenesis afterbrain insults: experimental approaches and translational research”Pharmacol Rev 2010, Vol. 62, 668-700; Shetty “Prospects of levetiracetamas a neuroprotective drug against status epilepticus, traumatic braininjury and stroke” Front. Neur. 2013, 4:172. Doi:10.3389/fneur.2013.00172) as it has displayed antiepileptogenic activityin the kindling model in mice and rats. It has also been suggested thatlevetiracetam inhibits glutamate release (Lee et al. “Levetiracetaminhibits glutamate transmission through presynaptic P/Q-type calciumchannels on the granule cells of the dentate gyrus” British Journal ofPharmacology 2009, Vol. 158, pp. 1753-1762).

Seletracetam and Brivaracetam, have been found to reduce the severity ofdystonia in the dt^(sz) mutant hamster model and may be helpful in somepatients suffering from dyskinetic and dystonic movement disorders(Hamann et al. “Brivaracetam and seletracetam, two new SV2A ligands,improve paroxysmal dystonia in the dt^(sz) mutant hamster” EuropeanJournal of Pharmacology 2008, Vol. 601, pp. 99-102).

Positive allosteric modulators of mGluR2 have emerged recently aspromising novel therapeutic approaches for the treatment of several CNSdisorders, including epilepsy, and some mGluR2 PAMs are currentlyundergoing clinical trials for the treatment of schizophrenia, andanxiety-depression (www.clinicaltrials.gov, see for instance:JNJ-40411813/ADX71149 by Addex Therapeutics and Janssen Pharmaceuticals,Inc.). The initial suggestion that drugs that dampen glutamatergictransmission may be efficacious in the treatment of epilepsy came fromacute non-clinical studies with mixed mGlu2/3 receptor agonists(Moldrich et al. “Glutamate metabotropic receptors as targets for drugtherapy in epilepsy” Eur J Pharmacol. 2003, Vol. 476, pp. 3-16).LY379268 and LY389795, two mGlu2/3 receptor agonists, were foundineffective in blocking MES seizures up to doses producing motorimpairment but were found effective in the 6 Hz model in adose-dependent manner (Barton et al. “Comparison of the effect ofglutamate receptor modulators in the 6 Hz and maximal electroshockseizure models” Epilepsy Research 2003, Vol. 56, pp. 17-26). Continuedadministration of an mGlu2/3 agonist paradoxically induced seizureactivity in long-term toxicology studies (Dunayevich et al. “Efficacyand tolerability of an mGlu2/3 agonist in the treatment of generalizedanxiety disorder” Neuropsychopharmacology. 2008, Vol. 33(7), pp.1603-10). This paradoxical effect may be related to agonist-inducedchanges in the sensitivity of the receptor system (tachyphylaxis), buthas not been reported however in preclinical models of epilepsy.Positive allosteric modulators, in contrast, modulate ongoingneurotransmission but are not directly stimulatory, thereby reducing therisk for tachyphylaxis.

Prior to seizure activity, increases in extracellular glutamate aremeasured in human hippocampus and the increase is sustained duringepileptogenic activity (During and Spencer “Extracellular hippocampalglutamate and spontaneous seizure in the conscious human brain” Lancet1993, Vol. 341(8861), pp. 1607-10), thus lending support to the ideathat a reduction in glutamate levels may be of benefit in the treatmentof epilepsy. In fact, during seizure activity glutamate levels increaseto potentially neurotoxic levels. Seizure activity results inprogressive structural damage in human brain inducing furtherabnormalities in glutamate metabolism (Petroff et al.“Glutamate-glutamine cycling in the epileptic human hippocampus”Epilepsia 2002, Vol. 43(7), pp. 703-10). Thus, an mGluR2 positiveallosteric modulator or an mGluR2 orthosteric agonist may be expected toprotect against seizure-induced neuronal damage.

WO2009/033704 and WO2010/130424 disclose mGluR2 positive allostericmodulators, uses thereof and processes for synthesizing the compounds.WO1997/18199 and WO2003/104217 disclose excitatory amino acid receptormodulator compounds that later were shown to have mGlu2/3 orthostericagonist activity (see for example Rorick-Kehn et al. (2007) The Journalof Pharmacology and Experimental therapeutics Vol. 321, No. 1, pp.308-317), further scientific and patent literature disclose additionalexamples of compounds having mGlu2/3 orthosteric agonist activity, andWO2008/150233 discloses compounds with mGluR2 allosteric activatoractivity.

Currently available anti-epileptic drugs do not solely affectglutamatergic transmission. Their mechanism of action is generallyconceptualized as altering the balance between excitatory(glutamate-mediated) and inhibitory (GABA-mediated) transmission(Johannessen Landmark “Antiepileptic drugs in non-epilepsy disorders:relations between mechanisms of action and clinical efficacy” CNS Drugs2008, Vol. 22(1), pp. 27-47).

A significant limiting factor in the use of SV2A ligands is tolerabilityand side-effect profile. For example the effective dose of levetiracetamfor partial onset seizures is dosed at 1000 mg, 2000 mg, and 3000 mg,given as twice-daily. The side effects reported for levetiracetaminclude aggressive or angry behavior, anxiety, change in personality,chills, cough or hoarseness, crying, depersonalization, diarrhea, drymouth, euphoria, fever, general feeling of discomfort or illness,headache, hyperventilation, irregular heartbeats, irritability, jointpain, loss of appetite, lower back or side pain, mental depression,muscle aches and pains, nausea, painful or difficult urination,paranoia, quick to react or overreact emotionally, rapidly changingmoods, restlessness, shaking, shivering, shortness of breath, sleepinessor unusual drowsiness, sore throat, stuffy or runny nose, sweating,trouble sleeping, unusual tiredness or weakness and vomiting. Thus,there is still a need to provide an effective treatment with a lowereffective dose of levetiracetam and a more favourable side effectprofile for the treatment of epilepsy and related disorders, not only inthe adult but also in the pediatric population.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Dose response for the 6 Hz 44 mA ED₅₀ determination for the Co.No. 2 and LEV alone and in combination.

FIG. 2: Isobolographic analysis for the combination of Co. No. 1 withlevetiracetam (LEV) in the 6 Hz (44 mA) assay. Initial ED₅₀ values(shown below) were determined for both Co. No. 1 and LEV (data points onx- and y-axes; filled diamonds). The theoretical line of additivityconnects the calculated ED₅₀ values for the two compounds (solid blackline). Theoretical ED₅₀ (+SEM) for three fixed dose ratio combinations(LEV:Co. No. 1) are plotted: 1:3—filled squares/solid black line,1:1—filled upward triangles/solid black line, and 3:1—filled downwardtriangles/solid black line. Experimental treatment doses were initiallyderived from theoretical values and adjusted according to observedeffects. Experimentally-determined ED₅₀ (+SEM) values for each fixeddose-ratio combination are also shown: 1:3′—open squares/dotted line,1:1′—open upward triangles/dotted line, and 3:1′—open downwardtriangles/dotted line. Comparisons between theoretical andexperimentally-determined ED₅₀ values were compared using a t-test(***P<0.001). N=8 per group. In FIG. 2, the ratio of LEV to Co. No. 1 isdepicted as follows:

LEV:Co. No. 1 ratio

 1:3 ED₅₀ (LEV) = 345 mg/kg (211-485)

 1:1 (intraperitoneally, i.p.)

 3:1 ED₅₀ (Co. No. 1) = 10.2 mg/kg (3.1-12.4)

 1:3′ (s.c.)

 1:1′

 3:1′

FIG. 3: Combination Studies for Co. No. 25-a with levetiracetam (LEV) inthe 6 Hz Assay (44 mA). At a dose of 10 mg/kg s.c., Co. No. 25-aincreases the potency of LEV, leading to an approximate 70-fold shift inthe ED₅₀. This suggests a positive pharmacodynamic relationship.

FIG. 4: Combination Studies for Co. No. 2-a with levetiracetam (LEV) inthe 6 Hz Assay (44 mA). At a dose of 10 mg/kg s.c., Co. No. 2-aincreases the potency of LEV, leading to an approximate 35-fold shift inthe ED₅₀. This suggests a positive pharmacodynamic relationship.

FIG. 5: Combination Studies for Co. No. 6-b with levetiracetam (LEV) inthe 6 Hz Assay (44 mA). At a dose of 10 mg/kg p.o., Co. No. 6-bincreases the potency of LEV, leading to an approximate 100-fold shiftin the ED₅₀. This suggests a positive pharmacodynamic relationship.

FIG. 6: Combination Studies for LY-404039 with levetiracetam (LEV) inthe 6 Hz Assay (44 mA). At a dose of 5 mg/kg s.c., LY-404039 increasesthe potency of LEV, leading to an approximate 27-fold shift in the ED₅₀.This suggests a positive pharmacodynamic relationship.

DESCRIPTION OF THE INVENTION

The present invention relates to a combination comprising

-   -   (a) a synaptic vesicle protein 2A (“SV2A”) ligand; and    -   (b) a positive allosteric modulator (“PAM”) of metabotropic        glutamatergic receptor subtype 2 (“mGluR2”) compound or a        pharmaceutically acceptable salt or a solvate thereof, or an        orthosteric agonist of metabotropic glutamatergic receptor        subtype 2 compound or a pharmaceutically acceptable salt or a        solvate thereof.

In a particular embodiment, the invention as described herein relates toa pharmaceutical combination, in particular a pharmaceutical combinationproduct, comprising

-   -   (a) a synaptic vesicle protein 2A (“SV2A”) ligand; and    -   (b) a positive allosteric modulator (“PAM”) of metabotropic        glutamatergic receptor subtype 2 (“mGluR2”) compound or a        pharmaceutically acceptable salt or a solvate thereof, or an        orthosteric agonist of metabotropic glutamatergic receptor        subtype 2 compound or a pharmaceutically acceptable salt or a        solvate thereof and    -   (c) at least one pharmaceutically acceptable carrier.

In a further embodiment, the invention relates to the combinationdescribed herein for use as a medicament.

A further embodiment of this invention relates to the use of thecombination described herein for the manufacture of a medicament or apharmaceutical product for the treatment or prevention of epilepsy andrelated disorders; neuropathic pain; migraine or resistant headache andbipolar and related disorders.

A further embodiment of this invention relates to the use of thecombination described herein for the manufacture of a medicament or apharmaceutical product for neuroprotection.

A further embodiment of this invention relates to the use of thecombination described herein for the manufacture of a medicament or apharmaceutical product for the prevention of epileptogenesis.

A further embodiment relates to the treatment or prevention of epilepsyand related disorders; neuropathic pain; migraine or resistant headache;and bipolar and related disorders of a subject comprising administeringconcurrently or sequentially to the subject in need thereof a synapticvesicle protein 2A (“SV2A”) ligand; and a positive allosteric modulator(“PAM”) of metabotropic glutamatergic receptor subtype 2 (“mGluR2”)compound or a pharmaceutically acceptable salt or a solvate thereof, oran orthosteric agonist of metabotropic glutamatergic receptor subtype 2compound or a pharmaceutically acceptable salt or a solvate thereof, inamounts that would be therapeutically effective when the SV2A ligand andmGluR2 compound are administered together.

A further embodiment relates to a combination as described herein forneuroprotection; or to a combination as described herein for use inneuroprotection.

A further embodiment relates to a combination as described herein forthe prevention of epileptogenesis; or to a combination as describedherein for use in the prevention of epileptogenesis.

In a further embodiment the invention relates to a method of treating orpreventing epilepsy and related disorders; neuropathic pain; migraine orresistant headache; bipolar and related disorders in patients comprisingadministering a fixed dose combination of

-   -   (a) a synaptic vesicle protein 2A (“SV2A”) ligand; and    -   (b) a positive allosteric modulator (“PAM”) of metabotropic        glutamatergic receptor subtype 2 (“mGluR2”) compound or a        pharmaceutically acceptable salt or a solvate thereof, or an        orthosteric agonist of metabotropic glutamatergic receptor        subtype 2 compound or a pharmaceutically acceptable salt or a        solvate thereof,        in amounts that would be therapeutically effective when the SV2A        ligand and mGluR2 compound are administered together.

In a further embodiment the invention relates to a method ofneuroprotection with a combination as defined herein.

In a further embodiment the invention relates to a method ofanti-epileptogenesis with a combination as defined herein.

A further embodiment relates to a method for the treatment or preventionof epilepsy and related disorders; neuropathic pain; migraine orresistant headache; bipolar and related disorders said method comprisingadministering a therapeutically effective amount of a combination or acombination product comprising

-   -   (a) a synaptic vesicle protein 2A (“SV2A”) ligand; and    -   (b) a positive allosteric modulator (“PAM”) of metabotropic        glutamatergic receptor subtype 2 (“mGluR2”) compound or a        pharmaceutically acceptable salt or a solvate thereof, or an        orthosteric agonist of metabotropic glutamatergic receptor        subtype 2 compound or a pharmaceutically acceptable salt or a        solvate thereof,    -   to a subject in need thereof, such as a warm-blooded animal, in        particular a human.

A further embodiment relates to a method of neuroprotection, said methodcomprising administering a therapeutically effective amount of acombination or a combination product comprising

-   -   (a) a synaptic vesicle protein 2A (“SV2A”) ligand; and    -   (b) a positive allosteric modulator (“PAM”) of metabotropic        glutamatergic receptor subtype 2 (“mGluR2”) compound or a        pharmaceutically acceptable salt or a solvate thereof, or an        orthosteric agonist of metabotropic glutamatergic receptor        subtype 2 compound or a pharmaceutically acceptable salt or a        solvate thereof,    -   to a subject in need thereof, such as a warm-blooded animal, in        particular a human.

A further embodiment relates to a method of anti-epileptogenesis, saidmethod comprising administering a therapeutically effective amount of acombination or a combination product comprising

-   -   (a) a synaptic vesicle protein 2A (“SV2A”) ligand; and    -   (b) a positive allosteric modulator (“PAM”) of metabotropic        glutamatergic receptor subtype 2 (“mGluR2”) compound or a        pharmaceutically acceptable salt or a solvate thereof, or an        orthosteric agonist of metabotropic glutamatergic receptor        subtype 2 compound or a pharmaceutically acceptable salt or a        solvate thereof,    -   to a subject in need thereof, such as a warm-blooded animal, in        particular a human.

In an additional embodiment, the present invention relates to apharmaceutical product or a commercial package comprising a combinationaccording to the invention as described herein, in particular togetherwith instructions, for simultaneous, separate or sequential use thereofin the treatment or prevention of epilepsy and related disorders;neuropathic pain; migraine or resistant headache bipolar; and relateddisorders.

In an additional embodiment, the present invention relates to apharmaceutical product or a commercial package comprising a combinationaccording to the invention as described herein, in particular togetherwith instructions, for simultaneous, separate or sequential use thereofin neuroprotection.

In an additional embodiment, the present invention relates to apharmaceutical product or a commercial package comprising a combinationaccording to the invention as described herein, in particular togetherwith instructions, for simultaneous, separate or sequential use thereofin anti-epileptogenesis.

In a further embodiment the invention relates to a combinationcomprising a quantity which is jointly therapeutically effective againstepilepsy and related disorders; neuropathic pain; migraine or resistantheadache; bipolar and related disorders; of

-   -   (a) a synaptic vesicle protein 2A (“SV2A”) ligand; and    -   (b) a positive allosteric modulator (“PAM”) of metabotropic        glutamatergic receptor subtype 2 (“mGluR2”) compound or a        pharmaceutically acceptable salt or a solvate thereof, or an        orthosteric agonist of metabotropic glutamatergic receptor        subtype 2 compound or a pharmaceutically acceptable salt or a        solvate thereof, and at least one pharmaceutically acceptable        carrier.

In a further embodiment the invention relates to a combinationcomprising a quantity which is jointly therapeutically effective asneuroprotectant, of

-   -   (a) a synaptic vesicle protein 2A (“SV2A”) ligand; and    -   (b) a positive allosteric modulator (“PAM”) of metabotropic        glutamatergic receptor subtype 2 (“mGluR2”) compound or a        pharmaceutically acceptable salt or a solvate thereof, or an        orthosteric agonist of metabotropic glutamatergic receptor        subtype 2 compound or a pharmaceutically acceptable salt or a        solvate thereof, and at least one pharmaceutically acceptable        carrier.

In a further embodiment the invention relates to a combinationcomprising a quantity which is jointly therapeutically effective in theprevention of epileptogenesis, of

-   -   (a) a synaptic vesicle protein 2A (“SV2A”) ligand; and    -   (b) a positive allosteric modulator (“PAM”) of metabotropic        glutamatergic receptor subtype 2 (“mGluR2”) compound or a        pharmaceutically acceptable salt or a solvate thereof, or an        orthosteric agonist of metabotropic glutamatergic receptor        subtype 2 compound or a pharmaceutically acceptable salt or a        solvate thereof, and at least one pharmaceutically acceptable        carrier.

In a further embodiment, the invention relates to the use of

-   -   (a) a synaptic vesicle protein 2A (“SV2A”) ligand; and    -   (b) a positive allosteric modulator (“PAM”) of metabotropic        glutamatergic receptor subtype 2 (“mGluR2”) compound or a        pharmaceutically acceptable salt or a solvate thereof, or an        orthosteric agonist of metabotropic glutamatergic receptor        subtype 2 compound or a pharmaceutically acceptable salt or a        solvate thereof,    -   for the preparation of a combination product according to the        present invention.

The (b) components of the combination of the invention are in generalreferred to herein as “mGluR2 compounds” or “mGluR2 PAM/agonistcompounds”, or “positive allosteric modulator of mGluR2/mGluR2orthosteric agonist compound” meaning that the compounds have mainlyactivity at the metabotropic glutamatergic receptor subtype 2, and arein particular selected from positive allosteric modulators (PAMs) ofmetabotropic glutamatergic receptor subtype 2, and orthosteric agonistsof metabotropic glutamatergic receptor subtype 2. A skilled person willbe familiar with the large homology of mGluR2 and mGluR3, due to whichsome mGluR2 orthosteric agonists also display activity as mGluR3orthosteric agonists. Such is the case for example, of(−)-(1R,4S,5S,6S)-4-amino-2-sulfonylbicyclo[3.1.0]-hexane-4,6-dicarboxylicacid (also known as LY-404,039 [CAS 635318-11-5]), with a K_(i)=149 nM(mGlu2 receptor) and K_(i)=92 nM (mGlu3 receptor), 100-fold selectivityfor mGlu2 and mGlu3 over mGlu4a, -6, -7a, and -8a, and no activity atmGlu1a and mGlu5a (Rorick-Kehn et al. (2007) The Journal of Pharmacologyand Experimental Therapeutics Vol. 321, No. 1, pp. 308-317). The term“mGluR2 compounds” or “mGluR2 PAM/agonist compounds”, or “positiveallosteric modulator of mGluR2/mGluR2 orthosteric agonist compound” doestherefore not exclude compounds displaying some other additional minoractivity in vitro or in vivo.

The mGluR2 PAM compounds of the combination of the invention are inparticular selected from those disclosed in WO2010/130424. A particularsubgroup of said compounds disclosed in WO2010/130424 can be defined bythe following Formula (I)

or a stereoisomeric form thereof; whereinR¹ is selected from the group consisting of (C₃₋₇cycloalkyl)C₁₋₃alkyl-,mono- or polyhaloC₁₋₄alkyl, and (C₁₋₄alkyl)-O—(C₁₋₄alkyl);R² is halo or polyhaloC₁₋₄alkyl;A is a covalent bond or a —CH₂—;L is selected from the radicals (a), (b) and (c):

whereinR^(3a) is selected from unsubstituted phenyl or phenyl substituted with1 or 2 halo substituents;R^(4a) is selected from the group of hydrogen, C₁₋₃alkyl and halo;or R^(3a)—C—R^(4a) together represent a radical of formula (a-1)

wherein R^(5a) is hydrogen or halo;R^(3b) is selected from the group of phenyl substituted with 1 or 2 halosubstituents, pyridinyl substituted with 1 or 2 halo substituents,unsubstituted pyrimidinyl and pyrimidinyl substituted with 1 or 2C₁₋₃alkyloxy substituents;or a pharmaceutically acceptable salt or a solvate thereof.

Thus, according to a particular embodiment of the invention, thepositive allosteric modulator (“PAM”) of metabotropic glutamatergicreceptor subtype 2 (“mGluR2”) compound is a compound of Formula (I) asdefined herein.

In a particular embodiment, the compounds of Formula (I) are as definedherein wherein

R¹ is selected from the group consisting of cyclopropylmethyl-,2,2,2-trifluoroethyl, and CH₃—O—CH₂—;

R² is chloro or CF₃;

A is a covalent bond or a —CH₂—;

L is selected from the radicals (a), (b) and (c):

whereinR^(3a) is selected from unsubstituted phenyl or phenyl substituted with1 or 2 fluoro substituents;R^(4a) is selected from the group of hydrogen, methyl and fluoro;or R^(3a)—C—R^(4a) together represent a radical of formula (a-1)

wherein R^(5a) is hydrogen or fluoro;R^(3b) is selected from the group of phenyl substituted with 1 or 2fluoro substituents, pyridinyl substituted with 1 or 2 fluorosubstituents, unsubstituted pyrimidinyl and pyrimidinyl substituted with1 or 2 methoxy substituents;or a pharmaceutically acceptable salt or a solvate thereof.

In a particular embodiment, the compounds of Formula (I) are as definedherein wherein

(i) when A is CH₂; and R² is trifluoromethyl; then

-   -   R¹ is cyclopropylmethyl-; and    -   L is selected from

(ii) when A is CH₂; and R₂ is chloro; then

-   -   R¹ is cyclopropylmethyl-; and    -   L is

(iii) when A is a covalent bond; and R² is trifluoromethyl; then

-   -   R¹ is cyclopropylmethyl; and    -   L is selected from

(iv) when A is a covalent bond and R² is Cl; then

-   -   (iv-a) R¹ is cyclopropylmethyl and L is

-   -   or    -   (iv-b) R¹ is 2,2,2-trifluoroethyl and L is selected from

(v) when A is CH₂ and R¹ is —CH₂—O—CH₃; thenR² is —CF₃ and L is

or a pharmaceutically acceptable salt or a solvate thereof.

The compounds of Formula (I) are disclosed in WO2010/130424 and may beprepared according to the processes described therein, which are herebyincorporated by reference in their totality.

Particular compounds of Formula (I) include

In an embodiment of the invention, the compound of Formula (I) is

or a pharmaceutically acceptable salt thereof, preferably ahydrochloride salt thereof.

In an additional embodiment of the invention, the compound of Formula(I) is

or a pharmaceutically acceptable salt thereof, preferably ahydrochloride salt thereof (.HCl).

The mGluR2 PAM compounds of the combination of the invention are also inparticular selected from those disclosed in WO2009/033704. Saidcompounds disclosed in WO2009/033704 can be defined by the followingFormula (I-A)

and the stereochemically isomeric forms thereof, whereinR¹ is C₁₋₆alkyl; or C₁₋₃alkyl substituted with C₃₋₇cycloalkyl, phenyl,or phenyl substituted with halo, trifluoromethyl or trifluoromethoxy;R² is halo, trifluoromethyl, C₁₋₃alkyl or cyclopropyl;R³ is hydrogen, fluoro, hydroxyl, hydroxyC₁₋₃alkyl, hydroxyC₁₋₃alkyloxy,fluoroC₁₋₃alkyl, fluoroC₁₋₃alkyloxy or cyano; andAr is unsubstituted phenyl; or phenyl substituted with n radicals R⁴,wherein n is 1, 2 or 3;R⁴ is selected from the group consisting of hydrogen, halo, C₁₋₃alkyl,hydroxyC₁₋₃alkyl, polyhaloC₁₋₃alkyl, cyano, hydroxyl, amino, carboxyl,C₁₋₃alkyloxyC₁₋₃alkyl, C₁₋₃alkyloxy, polyhaloC₁₋₃alkyloxy,C₁₋₃alkylcarbonyl, mono- and di(C₁₋₃alkyl)amino, and morpholinyl; ortwo vicinal R⁴ radicals taken together form a bivalent radical offormula—N═CH—NH—  (i),—CH═CH—NH—  (ii), or—O—CH₂—CH₂—NH—  (iii); orR³ and a R⁴ radical in ortho position taken together form a bivalentradical of formula—CH₂—O—  (iv), or—O—CH₂—  (v);

-   -   and the pharmaceutically acceptable salts and the solvates        thereof.

In a particular embodiment, the compounds of Formula (I-A) are asdefined herein wherein

R¹ is C₁₋₆alkyl; or C₁₋₃alkyl substituted with C₃₋₇cycloalkyl, phenyl,or phenyl substituted with halo, trifluoromethyl or trifluoromethoxy;

R² is halo, trifluoromethyl, C₁₋₃alkyl or cyclopropyl;

R³ is hydrogen, fluoro, hydroxyl, hydroxyC₁₋₃alkyl, hydroxyC₁₋₃alkyloxy,fluoroC₁₋₃alkyl, fluoroC₁₋₃alkyloxy or cyano; and

Ar is unsubstituted phenyl, or phenyl substituted with n radicals R⁴,wherein n is 1, 2 or 3;

R⁴ is selected from the group consisting of hydrogen, halo, C₁₋₃alkyl,hydroxyC₁₋₃alkyl, polyhaloC₁₋₃alkyl, cyano, hydroxyl, amino, carboxyl,C₁₋₃alkyloxyC₁₋₃alkyl, C₁₋₃alkyloxy, polyhaloC₁₋₃alkyloxy;C₁₋₃alkylcarbonyl, mono- and di(C₁₋₃alkyl)amino, and morpholinyl; ortwo vicinal R⁴ radicals taken together form a bivalent radical offormula—N═CH—NH—  (i),—CH═CH—NH—  (ii), or—O—CH₂—CH₂—NH—  (iii);and the pharmaceutically acceptable salts and solvates thereof.

In a particular embodiment, the compounds of Formula (I-A) are asdefined herein wherein

R¹ is C₁₋₆alkyl; or C₁₋₃alkyl substituted with C₃₋₇cycloalkyl, phenyl orphenyl substituted with halo, trifluoromethyl or trifluoromethoxy;

R² is halo, trifluoromethyl, C₁₋₃alkyl or cyclopropyl;

R³ is hydrogen, fluoro, hydroxyl, hydroxyC₁₋₃alkyl, hydroxyC₁₋₃alkyloxy,fluoroC₁₋₃alkyl, fluoroC₁₋₃alkyloxy or cyano; and

Ar is unsubstituted phenyl;

and the pharmaceutically acceptable salts and solvates thereof.

In an additional embodiment, the compounds of Formula (I-A) are asdefined herein wherein

R¹ is 1-butyl, 2-methyl-1-propyl, 3-methyl-1-butyl, (cyclopropyl)methylor 2-(cyclopropyl)-3-ethyl;

R³ is hydrogen, fluoro or cyano; and

Ar is unsubstituted phenyl;

and the pharmaceutically acceptable salts and solvates thereof.

In an additional embodiment, the compounds of Formula (I-A) are asdefined herein wherein

R¹ is 1-butyl, 3-methyl-1-butyl, (cyclopropyl)methyl or2-(cyclopropyl)-1-ethyl;

R² is chloro;

R³ is hydrogen or fluoro; and

Ar is unsubstituted phenyl;

and the pharmaceutically acceptable salts and the solvates thereof.

In a further embodiment, the compounds of Formula (I-A) are as definedherein wherein

R¹ is C₁₋₆alkyl; or C₁₋₃alkyl substituted with C₃₋₇cycloalkyl, phenyl,or phenyl substituted with halo, trifluoromethyl or trifluoromethoxy;

R² is halo, trifluoromethyl, C₁₋₃alkyl or cyclopropyl;

R³ is hydrogen, fluoro, hydroxyl, hydroxyC₁₋₃alkyl, hydroxyC₁₋₃alkyloxy,fluoroC₁₋₃alkyl, fluoroC₁₋₃alkyloxy or cyano; and

Ar is phenyl substituted with n radicals R⁴, wherein n is 1, 2, or 3;

R⁴ is selected from the group consisting of halo, C₁₋₃alkyl,hydroxyC₁₋₃alkyl, C₁₋₃alkyloxy, polyhaloC₁₋₃alkyloxy, C₁₋₃alkylcarbonyl,mono- and di(C₁₋₃alkyl)amino, and morpholinyl; or

two vicinal R⁴ radicals taken together form a bivalent radical offormula—N═CH—NH—  (i),—CH═CH—NH—  (ii), or—O—CH₂—CH₂—NH—  (iii); orR³ and a R⁴ radical in ortho position taken together form a bivalentradical of formula—CH₂—O—  (iv),—O—CH₂—  (v);and the pharmaceutically acceptable salts and solvates thereof.

In an additional embodiment, the compounds of Formula (I-A) are asdefined herein wherein

R¹ is 1-butyl, 2-methyl-1-propyl, 3-methyl-1-butyl, (cyclopropyl)methylor 2-(cyclopropyl)-1-ethyl;

R³ is hydrogen, fluoro or cyano; and

Ar is phenyl substituted with halo, trifluoromethyl, morpholinyl orhydroxyC₁₋₃alkyl;

and the pharmaceutically acceptable salts and solvates thereof.

In an additional embodiment, the compounds of Formula (I-A) are asdefined herein wherein

R¹ is 1-butyl, 3-methyl-1-butyl, (cyclopropyl)methyl or2-(cyclopropyl)-1-ethyl;

R² is chloro;

R³ is hydrogen or fluoro; and

Ar is phenyl substituted with at least one halo group;

and the pharmaceutically acceptable salts and solvates thereof.

In an additional embodiment, the compounds of Formula (I-A) are asdefined herein wherein

R¹ is 1-butyl, 3-methyl-1-butyl, (cyclopropyl)methyl or2-(cyclopropyl)-1-ethyl;

R² is chloro;

R³ is hydrogen or fluoro; and

Ar is phenyl substituted with at least two fluoro groups;

and the pharmaceutically acceptable salts and solvates thereof.

The compounds of Formula (I-A) are disclosed in WO2009/033704 and may beprepared according to the processes described therein, which are herebyincorporated by reference in their totality.

Particular compounds of Formula (I-A) include

and the pharmaceutically acceptable salts and the solvates thereof.

In an embodiment of the invention, the compound of Formula (I-A) is

or a pharmaceutically acceptable salt or a solvate thereof.

The mGluR2 PAM compounds of the combination of the invention are also inparticular selected from those disclosed in PCT/EP2014/068676. Saidcompounds disclosed in PCT/EP2014/068676 can be defined by the followingFormula (I-B)

and the stereochemically isomeric forms thereof, whereinR¹ is selected from the group consisting of C₁₋₆alkyl,(C₃₋₈cycloalkyl)C₁₋₃alkyl, and (C₁₋₃ alkyloxy)C₁₋₃ alkyl;each R² is independently selected from F, Cl, C₁₋₃alkyl, C₁₋₃alkyloxy,mono- or polyhaloC₁₋₃alkyl, and mono- or polyhaloC₁₋₃alkyloxy;n is an integer selected from 1, 2, and 3;and the pharmaceutically acceptable salts and the solvates thereof.

The mGluR2 PAM compounds of the combination of the invention are inparticular selected from compounds of Formula (I-B) as definedhereinabove, and stereoisomeric forms thereof, wherein R¹ is selectedfrom the group consisting of CH₃CH₂, CH₃CH₂CH₂, (cyclopropyl)methyl,(cyclobutyl)methyl, ethyloxymethyl and methyloxymethyl; and the rest ofvariables are as defined herein; and the pharmaceutically acceptablesalts and the solvates thereof.

In a further embodiment, the mGluR2 PAM compounds of the combination ofthe invention are in particular selected from compounds of Formula (I-B)as defined hereinabove, and stereoisomeric forms thereof, wherein R¹ isselected from the group consisting of CH₃CH₂, (cyclopropyl)methyl,(cyclobutyl)methyl and methyloxymethyl; and the rest of variables are asdefined herein; and the pharmaceutically acceptable salts and thesolvates thereof.

In a further embodiment, the mGluR2 PAM compounds of the combination ofthe invention are in particular selected from compounds of Formula (I-B)as defined hereinabove, and stereoisomeric forms thereof, wherein R¹ isselected from the group consisting of CH₃CH₂, (cyclopropyl)methyl,(cyclobutyl)methyl and ethyloxymethyl; and the rest of variables are asdefined herein; and the pharmaceutically acceptable salts and thesolvates thereof.

Thus, according to a particular embodiment of the invention, thepositive allosteric modulator (“PAM”) of metabotropic glutamatergicreceptor subtype 2 (“mGluR2”) compound is a compound of Formula (I-B) asdefined herein.

In an additional embodiment, the compounds of Formula (I-B) are asdefined herein, wherein

each R² is independently selected from F, Cl, CH₃, CH₃O and CF₃; and thepharmaceutically acceptable salts and the solvates thereof.

In a further embodiment, the compounds of Formula (I-B) are as definedherein having the Formula (I-Ba)

wherein the variables are as defined in Formula (I-B) herein, and thepharmaceutically acceptable salts and the solvates thereof.

In a further embodiment, the compounds of Formula (I-B) are as definedherein having the Formula (I-Bb)

wherein the variables are as defined in Formula (I-B) herein, and thepharmaceutically acceptable salts and the solvates thereof.

Particular compounds of formula (I-B) include

-   3-(Cyclopropylmethyl)-7-[1-(4-fluorophenoxy)ethyl]-8-(trifluoromethyl)[1,2,4]triazolo-[4,3-a]pyridine;-   3-(Cyclopropylmethyl)-7-[(1*R)-1-(4-fluorophenoxy)ethyl]-8-(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine;-   3-(Cyclopropylmethyl)-7-[(1*S)-1-(4-fluorophenoxy)ethyl]-8-(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine;-   3-(Cyclopropylmethyl)-7-[(1S)-1-(2,4-difluorophenoxy)ethyl]-8-(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine;-   3-(Cyclopropylmethyl)-7-[(1R)-1-(2,4-difluorophenoxy)ethyl]-8-(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine;-   3-(Cyclopropylmethyl)-7-[1-(2,4-difluorophenoxy)ethyl]-8-(trifluoromethyl)[1,2,4]triazolo-[4,3-a]pyridine;-   3-(Cyclopropylmethyl)-7-[(1S)-1-(3,5-difluorophenoxy)ethyl]-8-(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine;-   3-(Cyclopropylmethyl)-7-[(1S)-1-(3,4-difluorophenoxy)ethyl]-8-(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine;-   3-(Cyclopropylmethyl)-7-[(1S)-1-(2,3-difluorophenoxy)ethyl]-8-(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine;-   3-(Cyclopropylmethyl)-7-[(1S)-1-(2,5-difluorophenoxy)ethyl]-8-(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine;-   3-(Cyclopropylmethyl)-7-[(1S)-1-(2,6-difluorophenoxy)ethyl]-8-(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine;-   3-(Cyclopropylmethyl)-7-[(1S)-1-(4-fluoro-2-methoxyphenoxy)ethyl]-8-(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine;-   3-(Cyclobutylmethyl)-7-[1-(2,4-difluorophenoxy)ethyl]-8-(trifluoromethyl)[1,2,4]triazolo-[4,3-a]pyridine;-   7-[(1S)-1-(2-Chloro-4-methylphenoxy)ethyl]-3-(cyclopropylmethyl)-8-(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine;-   3-(Cyclopropylmethyl)-7-[(1S)-1-(4-fluoro-2-methylphenoxy)ethyl]-8-(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine;-   3-(Cyclopropylmethyl)-8-(trifluoromethyl)-7-[(1S)-1-(2,4,6-trifluorophenoxy)ethyl][1,2,4]triazolo[4,3-a]pyridine;-   7-[1-(2,4-Difluorophenoxy)ethyl]-3-(ethoxymethyl)-8-(trifluoromethyl)[1,2,4]triazolo-[4,3-a]pyridine;-   3-Ethyl-8-(trifluoromethyl)-7-[1-(2,4,6-trifluorophenoxy)ethyl][1,2,4]triazolo[4,3-a]pyridine;-   7-[1-(2,4-Difluorophenoxy)ethyl]-3-ethyl-8-(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine;-   3-(Cyclobutylmethyl)-7-[(1*R)-1-(2,4-difluorophenoxy)ethyl]-8-(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine;-   3-(Cyclobutylmethyl)-7-[(1*S)-1-(2,4-difluorophenoxy)ethyl]-8-(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine;-   3-(Ethoxymethyl)-8-(trifluoromethyl)-7-[(1*R)-1-(2,4,6-trifluorophenoxy)ethyl][1,2,4]triazolo[4,3-a]pyridine;-   3-(Ethoxymethyl)-8-(trifluoromethyl)-7-[(1*S)-1-(2,4,6-trifluorophenoxy)ethyl][1,2,4]triazolo[4,3-a]pyridine;-   7-[(1*S)-1-(2,4-Difluorophenoxy)ethyl]-3-(ethoxymethyl)-8-(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine;-   7-[(1*R)-1-(2,4-Difluorophenoxy)ethyl]-3-(ethoxymethyl)-8-(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine;-   7-[(1*R)-1-(2,4-Difluorophenoxy)ethyl]-3-ethyl-8-(trifluoromethyl)[1,2,4]triazolo-[4,3-a]pyridine;-   7-[(1*S)-1-(2,4-Difluorophenoxy)ethyl]-3-ethyl-8-(trifluoromethyl)[1,2,4]triazolo-[4,3-a]pyridine;-   7-[1-(2,4-Difluorophenoxy)ethyl]-3-propyl-8-(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine;-   3-Ethyl-8-(trifluoromethyl)-7-[(1*R)-1-(2,4,6-trifluorophenoxy)ethyl]-[1,2,4]triazolo-[4,3-a]pyridine;-   3-Ethyl-8-(trifluoromethyl)-7-[(1*S)-1-(2,4,6-trifluorophenoxy)ethyl]-[1,2,4]triazolo-[4,3-a]pyridine;-   7-[(1*R)-(2,4-difluorophenoxy)ethyl]-3-propyl-8-(trifluoromethyl)-[1,2,4]triazolo[4,3-a]pyridine;    and-   7-[(1*S)-(2,4-difluorophenoxy)ethyl]-3-propyl-8-(trifluoromethyl)-[1,2,4]triazolo[4,3-a]pyridine.

Included within the scope of this list are stereoisomeric forms, thepharmaceutically acceptable salts and the solvates thereof.

In an additional embodiment, the compound may be selected from

-   3-(Cyclopropylmethyl)-7-[(1S)-1-(2,4-difluorophenoxy)ethyl]-8-(trifluoromethyl)    [1,2,4]triazolo[4,3-a]pyridine hydrochloride salt.

The orthosteric agonists of mGluR2/mGluR2/3 of the combination of theinvention include, but are not limited to, for example, LY-404039;LY-2969822; LY-2934747; LY-379268; DCG-IV; LY-354740; LY-314582;LY-544344; LY-2140023; LY-181837; LY-389795; LY-446433; LY-450477;LY-395756; LY-566332; LY-541850; LY-2300559; LY-404040; LY-281223;LY-2979165; talaglumetad; MGS008; MGS0022; MGS0028; MGS0039;(−)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate;(+)-4-amino-2-sulfonylbicyclo[3.1.0]hexane-4,6-dicarboxylic acid;(+)-2-amino-4-fluorobicyclo-[3.1.0]hexane-2,6-dicarboxylic acid;1S,2R,5S,6S-2-amino-6-fluoro-4-oxobicyclo-[3.1.0]hexane-2,6-dicarboxylicacid;1S,2R,4S,5S,6S-2-amino-6-fluoro-4-hydroxybicyclo[3.1.0]hexane-2,6-dicarboxylicacid;1S,2R,3R,5S,6S-2-amino-3-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylicacid;1S,2R,3S,5S,6S-2-amino-6-fluoro-3-hydroxybicyclo[3.1.0]hexane-2,6-dicarboxylicacid; (+)-4-amino-2-sulfonylbicyclo-[3.1.0]hexane-4,6-dicarboxylic acid;(+)-2-amino-4-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid;1S,2R,5S,6S-2-amino-6-fluoro-4-oxobicyclo[3.1.0]hexane-2,6-dicarboxylicacid;1S,2R,4S,5S,6S-2-amino-6-fluoro-4-hydroxybicyclo[3.1.0]hexane-2,6-dicarboxylicacid;1S,2R,3R,5S,6S-2-amino-3-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylicacid; or1S,2R,3S,5S,6S-2-amino-6-fluoro-3-hydroxybicyclo-[3.1.0]hexane-2,6-dicarboxylicacid.

A particular group of mGluR2 agonists include LY-379268; DCG-IV;LY-354740; LY-404039; LY-2969822; LY-2934747; LY-544344; and LY-2140023.

The orthosteric agonists of metabotropic glutamatergic receptor subtype2 of the combination of the invention are in particular further selectedfrom those disclosed in WO1997/18199 and WO2003/104217, incorporatedherein in their entirety. Particular compounds disclosed therein are(−)-(1R,4S,5S,6S)-4-amino-2-sulfonylbicyclo[3.1.0]-hexane-4,6-dicarboxylicacid (also known as LY-404039)

or a salt or a solvate thereof, and(1R,4S,5S,6S)-4-[[(2S)-2-amino-4-(methylthio)-1-oxobutyl]amino]-2-thiabicyclo[3.1.0]hexane-4,6-dicarboxylicacid 2,2-dioxide (also known as LY-2140023 [CAS 635318-55-7])

or a salt or a solvate thereof, for example the monohydrate thereof.

The names of the compounds of the present invention were generatedaccording to the nomenclature rules agreed upon by the ChemicalAbstracts Service (C.A.S.) using Advanced Chemical Development, Inc.,software (ACD/Name product version 10.01.0.14105, October 2006). In caseof tautomeric forms, the name of the depicted tautomeric form of thestructure was generated. However it should be clear that the othernon-depicted tautomeric form is also included within the scope of thepresent invention.

As used herein, the notation “C₁₋₃alkyl”, “C₁₋₄alkyl” or “C₁₋₆alkyl” asa group or part of a group defines a saturated, straight or branched,hydrocarbon radical having from 1 to 3 or from 1 to 4 or from 1 to 6carbon atoms, such as methyl, ethyl, 1-propyl, 1-methylethyl, butyl,1-methylpropyl, 2-methyl-1-propyl, 1,1-dimethylethyl, 3-methyl-1-butyl,1-pentyl, 1-hexyl and the like.

The notation “C₃₋₇cycloalkyl” or “C₃₋₈cycloalkyl” as a group or part ofa group defines a saturated, cyclic hydrocarbon radical having from 3 to7 or from 3 to 8 carbon atoms, such as cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

The notation “halo” or “halogen” as used herein as a group or part of agroup refers to fluoro, chloro, bromo or iodo, with fluoro or chlorobeing preferred.

The notation “mono- and polyhaloC₁₋₃alkyl” or “mono- andpolyhaloC₁₋₄alkyl” shall denote C₁₋₃alkyl or C₁₋₄alkyl respectively, asdefined before, substituted with 1, 2, 3 or where possible with morehalo atoms as defined before.

Whenever the term “substituted” is used in the present invention, it ismeant, unless otherwise is indicated or is clear from the context, toindicate that one or more hydrogens, preferably from 1 to 3 hydrogens,more preferably from 1 to 2 hydrogens, more preferably 1 hydrogen, onthe atom or radical indicated in the expression using “substituted” arereplaced with a selection from the indicated group, provided that thenormal valency is not exceeded, and that the substitution results in achemically stable compound, i.e. a compound that is sufficiently robustto survive isolation to a useful degree of purity from a reactionmixture, and formulation into a therapeutic agent.

As used herein, unless otherwise noted, the term “antiepileptic agent”and the abbreviation “AED” will be used interchangeably with the term“anticonvulsant agent”, and as used herein, refer to an agent capable oftreating, inhibiting or preventing seizure activity or ictogenesis whenthe agent is administered to a subject or patient.

As used herein, unless otherwise noted, the term “synaptic vesicleprotein 2A ligand” and the abbreviation “SV2A ligand” will be usedinterchangeably. Examples of SV2A ligands include, but are not limitedto, the compounds included in the publications GB 1,039,113; GB1,309,692; EP 1 262 036; EP 1 806 339; WO 2001/062726; US 2002/094787;WO 2004/087658; WO 2005/121082; WO 2005/054188; WO 2006/128692; WO2006/128693; WO 2007/065595; WO 2008/132139, and WO 2008/132142; WO2011/047860; WO 2012/143116; and WO 2012/143117. Suitable particularexamples of SV2A ligands include, but are not limited to: levetiracetam,brivaracetam and seletracetam.

Therefore, in an embodiment of the invention, the SV2A ligand isselected from levetiracetam, brivaracetam and seletracetam.

In a particular embodiment, the SV2A ligand is levetiracetam.

In a particular embodiment, the SV2A ligand is brivaracetam.

Processes for the preparation of the above SV2A ligands are known fromthe literature and described for instance in EP 1 806 339; in EP 0 162036 and in GB 2 225 322 (levetiracetam); in WO 01/62726 (brivaracetam);and in WO 2005/121082 (seletracetam); such processes are herebyincorporated by reference in their totality.

In an additional embodiment, the combination according to the inventioncomprises (a) a SV2A ligand selected from levetiracetam or brivaracetam;and (b)

or a pharmaceutically acceptable salt thereof, preferably ahydrochloride salt thereof, or a solvate thereof.

In an additional embodiment, the pharmaceutical composition according tothe invention comprises (a) a pharmaceutically effective amount oflevetiracetam or brivaracetam; and (b) a pharmaceutically effectiveamount of

or a pharmaceutically acceptable salt thereof, preferably ahydrochloride salt thereof, or a solvate thereof.

In an additional embodiment, the combination according to the inventioncomprises (a) a pharmaceutically effective amount of levetiracetam orbrivaracetam; and (b) a pharmaceutically effective amount of

or a pharmaceutically acceptable salt, or a solvate thereof.

In an additional embodiment, the pharmaceutical composition according tothe invention comprises (a) a pharmaceutically effective amount oflevetiracetam or brivaracetam; and (b) a pharmaceutically effectiveamount of

or a pharmaceutically acceptable salt, or a solvate thereof.

In an additional embodiment, the combination according to the inventioncomprises (a) a pharmaceutically effective amount of levetiracetam orbrivaracetam; and (b) a pharmaceutically effective amount of

or a pharmaceutically acceptable salt, or a solvate thereof.

In an additional embodiment, the pharmaceutical composition according tothe invention comprises (a) a pharmaceutically effective amount oflevetiracetam or brivaracetam; and (b) a pharmaceutically effectiveamount of

or a pharmaceutically acceptable salt, or a solvate thereof.

In an additional embodiment, the combination according to the inventioncomprises (a) a pharmaceutically effective amount of levetiracetam orbrivaracetam; and (b) a pharmaceutically effective amount of

or a pharmaceutically acceptable salt thereof, in particular thehydrochloride salt thereof, or a solvate thereof.

In an additional embodiment, the pharmaceutical composition according tothe invention comprises (a) a pharmaceutically effective amount oflevetiracetam or brivaracetam; and (b) a pharmaceutically effectiveamount of

or a pharmaceutically acceptable salt thereof, in particular thehydrochloride salt thereof, or a solvate thereof.

In an additional embodiment, the combination according to the inventioncomprises (a) a pharmaceutically effective amount of levetiracetam orbrivaracetam; and (b) a pharmaceutically effective amount of LY-404039or a pharmaceutically acceptable salt thereof, in particular thehydrochloride salt thereof, or a solvate thereof.

In an additional embodiment, the pharmaceutical composition according tothe invention comprises (a) a pharmaceutically effective amount oflevetiracetam or brivaracetam; and (b) a pharmaceutically effectiveamount of LY-404039 or a pharmaceutically acceptable salt thereof, inparticular the hydrochloride salt thereof, or a solvate thereof.

In an additional embodiment, the combination according to the inventioncomprises (a) a pharmaceutically effective amount of levetiracetam orbrivaracetam; and (b) a pharmaceutically effective amount of LY-2140023or a pharmaceutically acceptable salt or a solvate thereof, inparticular the monohydrate thereof.

In an additional embodiment, the pharmaceutical composition according tothe invention comprises (a) a pharmaceutically effective amount oflevetiracetam or brivaracetam; and (b) a pharmaceutically effectiveamount of LY-2140023 or a pharmaceutically acceptable salt or a solvatethereof, in particular the monohydrate thereof.

The combination product of the present invention, in particular, thepharmaceutical composition according to the invention, is especiallyappropriate for the treatment of epilepsy and related disorders.

It will be appreciated that some of the mGluR2 compounds, in particularthe mGluR2 PAM/agonist compounds of the invention and theirpharmaceutically acceptable addition salts and solvates thereof maycontain one or more centres of chirality and exist as stereoisomericforms.

The term “compounds of the invention” as used herein, is meant toinclude the mGluR2 PAM compounds, in particular the compounds of Formula(I)/(I-A)/(I-B), and the mGluR2 agonist compounds as disclosed herein,and the salts and solvates thereof.

As used herein, any chemical formula with bonds shown only as solidlines and not as solid wedged or hashed wedged bonds, or otherwiseindicated as having a particular configuration (e.g. R, S) around one ormore atoms, contemplates each possible stereoisomer, or mixture of twoor more stereoisomers.

Hereinbefore and hereinafter, the terms “mGluR2 compound” and “mGluR2PAM/agonist compound” are meant to include the stereoisomers thereof andthe tautomeric forms thereof. The terms “stereoisomers”, “stereoisomericforms” or “stereochemically isomeric forms” hereinbefore or hereinafterare used interchangeably. The invention includes all stereoisomers ofthe compounds of the invention either as a pure stereoisomer or as amixture of two or more stereoisomers. Enantiomers are stereoisomers thatare non-superimposable mirror images of each other. A 1:1 mixture of apair of enantiomers is a racemate or racemic mixture. Diastereomers (ordiastereoisomers) are stereoisomers that are not enantiomers, i.e. theyare not related as mirror images. If a compound contains a double bond,the substituents may be in the E or the Z configuration. Substituents onbivalent cyclic (partially) saturated radicals may have either the cis-or trans-configuration; for example if a compound contains adisubstituted cycloalkyl group, the substituents may be in the cis ortrans configuration. Therefore, the invention includes enantiomers,diastereomers, racemates, E isomers, Z isomers, cis isomers, transisomers and mixtures thereof, whenever chemically possible. The meaningof all those terms, i.e. enantiomers, diastereomers, racemates, Eisomers, Z isomers, cis isomers, trans isomers and mixtures thereof areknown to the skilled person. The absolute configuration is specifiedaccording to the Cahn-Ingold-Prelog system. The configuration at anasymmetric atom is specified by either R or S. Resolved stereoisomerswhose absolute configuration is not known can be designated by (+) or(−) depending on the direction in which they rotate plane polarizedlight. For instance, resolved enantiomers whose absolute configurationis not known can be designated by (+) or (−) depending on the directionin which they rotate plane polarized light.

When a specific stereoisomer is identified, this means that saidstereoisomer is substantially free, i.e. associated with less than 50%,preferably less than 20%, more preferably less than 10%, even morepreferably less than 5%, in particular less than 2% and most preferablyless than 1%, of the other isomers. Thus, when a mGluR2 compound is forinstance specified as (R), this means that the compound is substantiallyfree of the (S) isomer; when a mGluR2 compound is for instance specifiedas E, this means that the compound is substantially free of the Zisomer; when a mGluR2 compound is for instance specified as cis, thismeans that the compound is substantially free of the trans isomer.

Some of the mGluR2 compounds may also exist in their tautomeric form.Such forms in so far as they may exist, although not explicitlyindicated in the above formula are intended to be included within thescope of the present invention.

It follows that a single compound may exist in both stereoisomeric andtautomeric forms.

For use in medicine, the salts of the compounds of this invention referto non-toxic “pharmaceutically acceptable salts” (salts of the compoundsof the present invention wherein the counterion is pharmaceuticallyacceptable). Other salts may, however, be useful in the preparation orpurification of compounds according to this invention or of theirpharmaceutically acceptable salts, and may encompass acids and baseswhich are non-pharmaceutically acceptable. All salts, whetherpharmaceutically acceptable or not, are included within the ambit of thepresent invention.

The pharmaceutically acceptable acid and base addition salts asmentioned hereinabove or hereinafter are meant to comprise thetherapeutically active non-toxic acid and base addition salt forms whichthe compounds of the invention are able to form. Suitablepharmaceutically acceptable salts of the compounds include acid additionsalts which may, for example, be formed by mixing a solution of thecompound with a solution of a pharmaceutically acceptable acid such asfor example, inorganic acids such as hydrohalic acids, e.g. hydrochloricor hydrobromic acid, sulfuric, nitric, phosphoric and the like acids; ororganic acids such as, for example, acetic, propanoic, hydroxyacetic,lactic, pyruvic, oxalic (i.e. ethanedioic), malonic, succinic (i.e.butanedioic acid), maleic, fumaric, malic, tartaric, citric,methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic,cyclamic, salicylic, p-aminosalicylic, pamoic and the like acids.Conversely, said salt forms can be converted by treatment with anappropriate base into the free base form. Furthermore, where thecompounds of the invention carry an acidic moiety, suitablepharmaceutically acceptable salts thereof may include organic andinorganic bases. Appropriate base salt forms comprise, for example, theammonium salts, the alkali and alkaline earth metal salts, e.g. thelithium, sodium, potassium, magnesium, calcium salts and the like, saltswith organic bases, e.g. primary, secondary and tertiary aliphatic andaromatic amines such as methylamine, ethylamine, propylamine,isopropylamine, the four butylamine isomers, dimethylamine,diethylamine, diethanolamine, dipropylamine, diisopropylamine,di-n-butylamine, pyrrolidine, piperidine, morpholine, trimethylamine,triethylamine, tripropylamine, quinuclidine, pyridine, quinoline andisoquinoline; the benzathine, N-methyl-D-glucamine, hydrabamine salts,and salts with amino acids such as, for example, arginine, lysine andthe like. Conversely, the salt form can be converted by treatment withacid into the free acid form.

The term “solvate” comprises the solvent addition forms as well as thesalts thereof, which the compounds of Formula (I) are able to form.Examples of such solvent addition forms are e.g. hydrates, alcoholatesand the like.

Preparation of the Compounds of Formula (I-B)

The compounds of Formula (I-B) according to the invention can generallybe prepared by a succession of steps, each of which is known to theskilled person. In particular, the compounds can be prepared accordingto the following synthesis methods.

The compounds of Formula (I-B) may be synthesized in the form of racemicmixtures of enantiomers which can be separated from one anotherfollowing art-known resolution procedures. The racemic compounds ofFormula (I-B) may be converted into the corresponding diastereomericsalt forms by reaction with a suitable chiral acid. Said diastereomericsalt forms are subsequently separated, for example, by selective orfractional crystallization and the enantiomers are liberated therefromby alkali. An alternative manner of separating the enantiomeric forms ofthe compounds of Formula (I-B) involves liquid chromatography orsupercritical fluid chromatography (SFC) using a chiral stationaryphase. Said pure stereochemically isomeric forms may also be derivedfrom the corresponding pure stereochemically isomeric forms of theappropriate starting materials, provided that the reaction occursstereospecifically.

A. Preparation of the Final Compounds of Formula (I-B)

Final compounds according to Formula (I-B), can be prepared by reactingan intermediate compound of Formula (II) with a compound of Formula(III) according to reaction scheme (1), a reaction that is performedunder classical Mitsunobu conditions. The reaction is preferablyconducted with a phosphine and an azodicarboxylic ester or amide intetrahydrofuran, 1,4-dioxane, diethyl ether, toluene, benzene,dichloromethane, or mixtures thereof, at −30 to 150° C., under thermalheating or microwave irradiation. Phosphines often used aretriphenylphosphine and tributylphosphine which are usually combined withdimethyl azodicarboxylate, diethyl azodicarboxylate, diisopropylazodicarboxylate, di-(4-chlorobenzyl) azodicarboxylate, dibenzylazodicarboxylate, di-tert-butyl azodicarboxylate, azodicarboxylic acidbis-(dimethylamide), azodicarboxylic acid dipiperidine, orazodicarboxylic acid dimorpholine. In reaction scheme (1), all variablesare as defined in Formula (I-B)

B. Preparation of the IntermediatesExperimental Procedure 2

Intermediate compounds according to Formula (II) can be prepared bysubjecting an intermediate of Formula (IV) to conditions that are knownto those skilled in the art. This is illustrated in reaction scheme (2)wherein all variables are defined as mentioned hereinabove. Methodsaccomplishing these transformations are well known to those skilled inthe art. Treatment of the aldehyde of formula (IV) with anorganometallic such as methyl lithium or methyl magnesium bromide givesa compound of formula (II). A suitable solvent for this reaction is anether such as tetrahydrofuran and the reaction is usually carried out ata temperature between −78° C. and 40° C. In reaction scheme (2), allvariables are defined as in Formula (I-B).

Experimental Procedure 3

Intermediate compounds according to Formula (IV) can be prepared byreacting an intermediate of Formula (V) under dihydroxylation andoxidative cleavage conditions that are known to those skilled in the artand can be realized for example with oxone, osmium tetroxide. Theprocess may be carried out optionally in a solvent such as 1,4-dioxane,water and generally at temperatures between about −100° C. and about100° C. A summary of such methods is found in “Comprehensive OrganicTransformations”, VCH Publishers, (1989), R. C. Larock, pp. 595-596.This is illustrated in reaction scheme (3) wherein all variables aredefined as mentioned hereinabove.

Experimental Procedure 4

Intermediate compounds according to Formula (V) can be prepared bycoupling reactions, such as Stille or Suzuki reactions of anintermediate of Formula (VI) with a compound of Formula (VII) underconditions that are known to those skilled in the art. The process maybe carried out optionally in a solvent such as 1,4-dioxane, water andgenerally at temperatures between about r.t. and about 200° C. in thepresence of a base. This is illustrated in reaction scheme (4) whereinall variables are defined as mentioned hereabove, wherein M istrialkyltin, boronic acid or boronate ester, and a palladium catalystand halo is chloro, bromo or iodo.

Experimental Procedure 5

Intermediate compounds according to Formula (VI) can be preparedfollowing art known procedures by cyclization of an intermediatecompound of Formula (VIII) in the presence of a halogenating agent suchas for example phosphorus (V) oxychloride (POCl₃) in a suitable solventsuch as, for example, dichloroethane, stirred under microwaveirradiation, for a suitable period of time that allows the completion ofthe reaction, as for example 5 min at a temperature between 140-200° C.In reaction scheme (5), R¹ is defined as in Formula (I-B) and halo ischloro, bromo or iodo.

Experimental Procedure 6

Intermediate compounds according to Formula (VIII) can be prepared byart known procedures by reaction of a hydrazine intermediate of Formula(IX) with acid halides of Formula (X). The reaction can be carried outusing an inert-solvent, such as for example DCM, in the presence of abase such as for example triethylamine, for example at r.t. for asuitable period of time that allows completion of the reaction, forexample 20 min. In reaction scheme (6), R¹ is defined as in Formula(I-B).

Experimental Procedure 7

Intermediate compounds according to Formula (IX) can be prepared byreacting an intermediate compound of Formula (XI) with hydrazineaccording to reaction scheme (7), a reaction that is performed in asuitable reaction-inert solvent, such as, for example, ethanol, THF or1,4-dioxane under thermal conditions such as, for example, heating thereaction mixture for example at 160° C. under microwave irradiation for30 min or classical thermal heating at 70° C. for 16 h. In reactionscheme (7), halo is chloro, bromo or iodo.

Experimental Procedure 8

Intermediate compounds according to Formula (XI) can be prepared byreacting an intermediate compound of Formula (XII) with benzyl alcoholaccording to reaction scheme (8), a reaction that is performed in asuitable reaction-inert solvent, such as, for example,N,N-dimethylformamide in the presence of a suitable base, such as forexample sodium hydride at r.t. for a suitable period of time that allowsthe completion of the reaction, such as for example 1 h. In reactionscheme (8), halo is chloro, bromo or iodo.

Experimental Procedure 9

Intermediate compounds of Formula (XII), can be prepared by reacting anintermediate of Formula (XIII), with a suitable trifluoromethylatingagent, such as for example fluorosulfonyl(difluoro)acetic acid methylester, according to reaction scheme (9). This reaction is performed in asuitable reaction-inert solvent such as, for example,N,N-dimethylformamide in the presence of a suitable coupling agent suchas for example, copper(I) iodide, under thermal conditions such as, forexample, heating the reaction mixture for example at 160° C. undermicrowave irradiation for 45 min. In reaction scheme (9), halo ischloro, bromo or iodo.

The starting materials according to Formulae (II), (VII), (X) or (XIII)are compounds that are either commercially available or may be preparedaccording to conventional reaction procedures generally known to thoseskilled in the art.

As used herein, the term “composition” is intended to encompass aproduct comprising the specified ingredients in the specified amounts,as well as any product which results, directly or indirectly, fromcombinations of the specified ingredients in the specified amounts.

As used herein, the term “subject” refers to an animal, preferably amammal, most preferably a human adult, child or infant, who is or hasbeen the object of treatment, observation or experiment.

The term “therapeutically effective amount” as used herein, means thatamount of active compound or pharmaceutical agent that elicits thebiological or medicinal response in a tissue system, animal or humanthat is being sought by a researcher, veterinarian, medical doctor orother clinician, which includes alleviation of one or more of thesymptoms of the disease or disorder being treated; and/or reduction ofthe severity of one or more of the symptoms of the disease beingtreated.

The combination of compounds (a) SV2A ligand and (b) positive allostericmodulator (“PAM”) of metabotropic glutamatergic receptor subtype 2(“mGluR2”) or a pharmaceutically acceptable salt or a solvate thereof,or orthosteric agonist of metabotropic glutamatergic receptor subtype 2or a pharmaceutically acceptable salt or a solvate thereof, whether thecompounds (a) and (b) are given simultaneously, separately orsequentially, may be beneficial compared to the effect of the compounds(a) or (b) administered alone. In particular, there may be at least onebeneficial effect, e.g. a mutual enhancement of the effect of thecompounds (a) and (b), a more than additive effect, in particular asynergic effect; additional advantageous effects, include for example, asignificantly reduced effective dose for the combination of (a) and (b);a further therapeutic effect not observed for any of the compounds (a)or (b) alone, a more beneficial side effect profile, or a combinedtherapeutic effect in a non-effective dosage of one or both of (a) and(b).

As defined herein, the term “fixed-dose ratio of (a) synaptic vesicleprotein 2A ligand to (b) compound of Formula (I) of 1:1, calculated onthe ED₅₀ values of the individual compounds (a) and (b)” refers tocompositions comprising compounds (a) and (b) in a dose corresponding to50% of the respective ED₅₀ dose of the individual compounds (a) and (b)or a multiple of this fixed-dose ratio. The term “fixed-dose ratio of(a) synaptic vesicle protein 2A ligand:(b) compound of Formula (I) of3:1, calculated on the ED₅₀ values of the individual compounds (a) and(b)” refers to compositions comprising (b) the compound of Formula (I)in a dose corresponding to 75% of the respective ED₅₀ dose and compound(a) in a dose corresponding to 25% of the respective ED₅₀ dose ofcompound (a) or a multiple of this fixed-dose ratio, and so on.

Thus, in another embodiment of the invention, (a) the SV2A ligand and(b) the compound of Formula (I) are present in the pharmaceuticalcomposition in a fixed-dose ratio of (a):(b) of about 1:10 to about10:1, preferably about 1:5 to about 5:1, more preferably about 1:3 toabout 3:1, in another embodiment of about 1:1 to about 3:1; in analternate embodiment of 1:3; in yet another embodiment of 1:1; furtherembodiment of 3:1; wherein the fixed-dose ratio is calculated on theED₅₀ values of the individual compounds (a) and (b).

Wherein the present invention is directed to co-therapy or combinationtherapy, comprising administration of (a) synaptic vesicle protein 2A(“SV2A”) ligand; and (b) a mGluR2 PAM/agonist compound, in particular acompound of Formula (I)/(I-A)/(I-B) as defined herein, pharmaceuticallyor therapeutically effective amount shall mean that amount of thecombination of agents taken together so that the combined effect elicitsthe desired biological or medicinal response. For example, thetherapeutically effective amount of co-therapy comprising administrationof (a) a SV2A ligand as defined herein and (b) a mGluR2 PAM/agonistcompound, in particular a compound of Formula (I)/(I-A)/(I-B) as definedherein would be the amount of the (a) a SV2A ligand as defined hereinand the amount of (b) a mGluR2 PAM/agonist compound, in particularcompound of Formula (I)/(I-A)/(I-B) that when taken together orsequentially have a combined effect that is therapeutically effective.Further, it will be recognized by one skilled in the art that in thecase of co-therapy with a therapeutically effective amount, as in theexample above, the amount of the mGluR2 PAM/agonist compound, inparticular the compound of Formula (I)/(I-A)/(I-B), and/or the amount ofthe suitable SV2A ligand individually may or may not be therapeuticallyeffective.

The present invention provides methods of prevention or treatmentcomprising administering to a subject in need thereof, co-therapy with atherapeutically effective amount of a SV2A ligand and a therapeuticallyeffective amount of a mGluR2 PAM/agonist compound, in particular acompound of formula (I)/(I-A)/(I-B), as described herein. In order toaccomplish this objective the compounds or compositions of thisinvention must be used in the correct therapeutically effective amountor dose, as described below.

Optimal dosages and schedules to be administered may be readilydetermined by those skilled in the art, and will vary with theparticular compound used, the mode of administration, the strength ofthe preparation, the mode of administration, and the advancement of thedisease condition. In addition, factors associated with the particularpatient being treated, including patient age, weight, diet and time ofadministration, will result in the need to adjust dosages.

One skilled in the art will recognize that a therapeutically effectivedosage of the compounds of the present invention can include repeateddoses within a prolonged treatment regimen that will yield clinicallysignificant results.

The amounts of the mGluR2 PAM/agonist compound, in particular of thecompound of Formula (I)/(I-A)/(I-B), in the combinations of theinvention that are administered on a daily basis may vary from about0.01 to about 2000 mg. Examples of daily amounts of the compound ofFormula (I)/(I-A)/(I-B) are 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0,15.0, 25.0, 50.0, 100, 150, 200, 250, 300, 400, 500, 750 and 1000milligrams for the symptomatic adjustment of the dosage to the patientto be treated. An effective amount of the drug is ordinarily supplied ata dosage level of from about 0.01 mg/kg to about 150.0 mg/kg of bodyweight per day or any range therein. Preferably, the range is from about0.1 to about 100.0 mg/kg of body weight per day, more preferably, fromabout 0.5 mg/kg to about 50 mg/kg, more preferably, from about 1.0 toabout 25.0 mg/kg of body weight per day. The compounds may beadministered on a regimen of 1, 2, 3 or 4 times per day. The amounts ofSV2A ligand that are administered on a daily basis may vary from about0.01 to about 7000 mg, preferably will be between 250 and 5000 mg andmore preferably will be between 500 and 3000 mg. Examples of dailyamount of the SV2A ligand are 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100,150, 200, 250, 500, 750, 1000, 1500 and 3000 milligrams for thesymptomatic adjustment of the dosage of the patient to be treated. Aneffective amount of the drug is ordinarily supplied at a dosage level offrom about 0.01 mg/kg to about 150.0 mg/kg of body weight per day or anyrange therein. Preferably, the range is from about 0.1 to about 100.0mg/kg of body weight per day, more preferably, from about 0.5 mg/kg toabout 50 mg/kg, more preferably, from about 1.0 to about 25.0 mg/kg ofbody weight per day. The compounds may be administered on a regimen of1, 2, 3 or 4 times per day. All amounts mentioned in this and thefollowing paragraphs refer to the free form (i.e. non-salt form). Theabove values represent free-form equivalents, i.e. quantities as if thefree form would be administered. If salts are administered the amountsneed to be calculated in function of the molecular weight ratio betweenthe salt and the free form.

The above mentioned daily doses are calculated for an average bodyweight of about 70 kg and should be recalculated in case of pediatricapplications, or when used with patients with a substantially divertingbody weight.

The dosages may be presented as one, two, three or four or moresub-doses administered at appropriate intervals throughout the day. Thedosage used preferably corresponds to the daily amount of the mGluR2PAM/agonist compound, in particular of the compound of Formula(I)/(I-A)/(I-B), or of the SV2A ligand, mentioned above, or a sub-dosethereof, such as ½, ⅓, ¼ thereof. A dosage form may contain the mGluR2PAM/agonist compound, in particular the compound (I)/(I-A)/(I-B), or theSV2A ligand or both together, in an amount equal to the ranges orquantities mentioned in the previous paragraphs, for example a dosageform may contain 10 mg, 25 mg, 50 mg, 100 mg, 150 mg, or 200 mg ofmGluR2 PAM/agonist compound, in particular of compound (I)/(I-A)/(I-B),10 mg, 25 mg, 50 mg, 100 mg or 250 mg, of SV2A ligand, either inseparate formulations or in a combined formulation. In one embodiment,the mGluR2 PAM/agonist compound, in particular the compound of Formula(I)/(I-A)/(I-B), is administered once daily (q.d.), in particular as onedose per day, and the SV2A ligand is administered once or twice daily(q.d. or b.i.d.), in particular as one or as two doses per day. In theinstance where both compounds are to be administered once daily, thiscan be accomplished by administering two separate doses, one with themGluR2 PAM/agonist compound, in particular the compound of Formula(I)/(I-A)/(I-B), one with the SV2A ligand, or by administering acombined dose containing the mGluR2 PAM/agonist compound, in particularthe compound of Formula (I)/(I-A)/(I-B), and SV2A ligand.

The combinations of the invention may be administered once, twice,three, four, or if desired, multiple times daily. In one embodiment, thecombination is administered once daily. In another embodiment, thecombination is administered twice daily, or three times per day.Administration of dosages may be by separate dosage forms, i.e. dosageforms only containing mGluR2 PAM/agonist compound, in particularcompound of Formula (I)/(I-A)/(I-B), or only SV2A ligand; or by combineddosage forms containing active ingredients mGluR2 PAM/agonist compound,in particular compound of Formula (I)/(I-A)/(I-B), and SV2A ligand.Also, a mix of using a combined dosage form and separate dosage formscan be used. Dosage forms that can be administered are describedhereinafter, oral dosage forms, in particular tablets or capsules beingpreferred.

Active ingredients may be formulated in pharmaceutical compositionseither separately or as a combined pharmaceutical composition. In thelatter instance, there is provided a pharmaceutical compositioncomprising a therapeutically effective amount of the mGluR2 PAM/agonistcompound, in particular of the compound of Formula (I)/(I-A)/(I-B), or apharmaceutically acceptable salt thereof, and the SV2A ligand, theforegoing being as specified herein, and a pharmaceutically acceptablecarrier.

In a further aspect, this invention relates to a process for preparing apharmaceutical composition as specified herein, which comprisesintimately mixing a pharmaceutically acceptable carrier with atherapeutically effective amount of the mGluR2 PAM/agonist compound, inparticular of the compound of Formula (I)/(I-A)/(I-B), or apharmaceutically acceptable salt or a solvate thereof, and atherapeutically effective amount of at least one SV2A ligand.

The combinations provided herein may also be formulated as a combinedpreparation for simultaneous, separate or sequential use in theprevention or treatment of epilepsy and related disorders; neuropathicpain; migraine or resistant headache; bipolar and related disorders; inneuroprotection; or in the prevention of epileptogenesis. In such acase, the mGluR2 PAM/agonist compound, in particular the compound ofFormula (I)/(I-A)/(I-B), is formulated in a pharmaceutical compositioncontaining other pharmaceutically acceptable excipients, and the SV2Aligand is formulated separately in a pharmaceutical compositioncontaining other pharmaceutically acceptable excipients. Conveniently,these separate pharmaceutical compositions can be part of a kit forsimultaneous, separate, or sequential use.

The individual components of the combination of the present inventioncan be administered simultaneously or separately at different timesduring the course of therapy or concurrently in divided or singlecombination forms.

Therefore, the mGluR2 PAM/agonist compounds, in particular the compoundsof Formula (I)/(I-A)/(I-B), and the SV2A ligand, individually orcombined, may be formulated into various pharmaceutical compositionssuitable for administration purposes. In these, a therapeuticallyeffective amount of the particular compound, or of both two compounds,is combined with a pharmaceutically acceptable carrier, which carriermay take a wide variety of forms depending on the form of preparationdesired for administration. Pharmaceutical compositions may be preparedas medicaments to be administered orally, parenterally (includingsubcutaneously (s.c.), intramuscularly (i.m.), and intravenously(i.v.)), rectally, transdermally, bucally, or nasally. Thepharmaceutical compositions may also be prepared to be administereddirectly to the nervous system by routes including, but not limited to,intracerebral, intraventricular, intracerebroventricular, intrathecal,intracisternal, intraspinal and/or peri-spinal route by delivery viaintracranial or intravertebral needles and/or catheters with or withoutpump devices. Suitable compositions for oral administration includepowders, granulates, aggregates, tablets, compressed or coated pills,dragees, sachets, hard or gelatin capsules, syrups and suspensions.Suitable compositions for parenteral administration include aqueous ornon-aqueous solutions or emulsions, while for rectal administrationsuitable compositions for administration include suppositories with ahydrophilic or hydrophobic vehicle. For topical administration suitabletransdermal delivery systems can be used and for nasal delivery suitableaerosol delivery systems can be used.

For example, in preparing the compositions for oral administration, anyof the usual pharmaceutical media may be employed such as, for example,water, glycols, oils, alcohols and the like in the case of oral liquidcompositions such as suspensions, syrups, elixirs, emulsions andsolutions; or solid carriers such as starches, sugars, kaolin,lubricants, binders, disintegrating agents and the like in the case ofsolid compositions. For parenteral compositions, the carrier willusually comprise sterile water, at least in large part, though otheringredients, such as solubilizers, emulsifiers or further auxiliariesmay be added thereto. Injectable solutions may be prepared in which thecarrier comprises saline solution, glucose solution or a mixture ofboth. Injectable suspensions may also be prepared in which caseappropriate liquid carriers, suspending agents and the like may beemployed. Also included are solid form preparations intended to beconverted, shortly before use, to liquid form preparations such aspowders for reconstitution. In the compositions suitable forpercutaneous administration, the carrier optionally comprises a skinpenetration enhancing agent and/or a wetting agent, optionally combinedwith suitable skin-compatible additives in minor proportions. The mGluR2PAM/agonist compound, in particular the compound of Formula(I)/(I-A)/(I-B), or SV2A ligand, or combinations thereof, may also beadministered via oral inhalation or insufflation by formulations suitedfor this type of administration such as a solution, a suspension or adry powder. Suitable pharmaceutical compositions for administration inthe form of aerosols or sprays are, for example, suspensions of themGluR2 PAM/agonist compound, in particular of the compound of Formula(I)/(I-A)/(I-B), or SV2A ligand, or both, in a pharmaceuticallyacceptable liquid carrier, such as ethanol or water, or a mixturethereof. If required, the formulation can also additionally containother pharmaceutical auxiliaries such as surfactants, emulsifiers andstabilizers as well as a propellant. Such a preparation customarilycontains the active compound in a concentration from approximately 0.1to 50%, in particular from approximately 0.3 to 3% by weight.

The pharmaceutical compositions may contain the active ingredient mGluR2PAM/agonist compound, in particular compound of Formula (I)/(I-A)/(I-B),or the SV2A ligand, or both combined in a concentration of about 0.1% toabout 50%, or about 1% to about 30%, or about 3% to about 20%, or about5% to about 20%, all percentages being by weight, wherein the total ofall components in said pharmaceutical compositions does not exceed 100%.In the compositions containing both two compounds mGluR2 PAM/agonistcompound, in particular compound of Formula (I)/(I-A)/(I-B), and SV2Aligand, the mGluR2 PAM/agonist compound, in particular the compound ofFormula (I)/(I-A)/(I-B), is present in a concentration of about 0.1% toabout 50%, or about 1% to about 30%, or about 3% to about 20%, or about5% to about 20%; and the SV2A ligand is present in a concentration ofabout 3% to about 50%, or about 5% to about 50%, or about 10% to about50%, or about 10% to about 40%, or about 10% to about 30%, wherein thetotal of all components in said pharmaceutical compositions does notexceed 100%.

The pharmaceutical compositions may be conveniently presented in unitdosage form for ease of administration and uniformity of dosage.Examples include tablets (including scored or coated tablets), capsules,pills, suppositories, powder packets, wafers, injectable solutions orsuspensions and the like, and segregated multiples thereof. Of interestare solid dosage forms for oral administration such as tablets orcapsules.

The solid dosage forms in unit dose form may be packed in any knownpackage, blister packs being preferred, in particular for tablets andcapsules. Where the mGluR2 PAM/agonist compound, in particular thecompound of Formula (I)/(I-A)/(I-B), and SV2A ligand are formulatedseparately, they could be packed in separate blisters, but one blistercould as well comprise unit dose forms of the mGluR2 PAM/agonistcompound, in particular of the compound of Formula (I)/(I-A)/(I-B), andof the SV2A ligand, for example one row with units of mGluR2 PAM/agonistcompound, in particular of compound of Formula (I)/(I-A)/(I-B), andanother with SV2A ligand. Other possibilities may be possible as well.

The combinations of this invention may be used to treat or preventepilepsy and related disorders; neuropathic pain; migraine or resistantheadache; and bipolar and related disorders; or they may be used as aneuroprotectant or to prevent epileptogenesis.

As used herein, the term “treatment” is intended to refer to allprocesses, wherein there may be a slowing, interrupting, arresting orstopping of the progression of a disease or an alleviation of symptoms,but does not necessarily indicate a total elimination of all symptoms

As used herein, unless otherwise noted, the terms “epilepsy and relateddisorders” or “epilepsy or related disorder” shall mean any disorder inwhich a subject (preferably a human adult, child or infant) experiencesone or more seizures and/or tremors. Suitable examples include, but arenot limited to, epilepsy (including, but not limited to,localization-related epilepsies, generalized epilepsies, epilepsies withboth generalized and local seizures, and the like), partial-onsetseizures with or without generalization, myoclonic seizures, primarygeneralized tonic-clonic seizures in particular in patients withidiopathic generalized epilepsy, seizures associated with Lennox-Gastautsyndrome, seizures as a complication of a disease or condition (such asseizures associated with encephalopathy, phenylketonuria, juvenileGaucher's disease, Lundborg's progressive myoclonic epilepsy, stroke,head trauma, stress, hormonal changes, drug use or withdrawal, alcoholuse or withdrawal, sleep deprivation, fever, infection, and the like),status epilepticus (convulsive or non convulsive), essential tremor,restless limb syndrome, and the like. Preferably, the disorder isselected from epilepsy (regardless of type, underlying cause or origin),essential tremor or restless limb syndrome. More preferably, thedisorder is epilepsy (regardless of type, underlying cause or origin) oressential tremor. A particular example of epilepsy is refractoryepilepsy, also referred to as treatment or therapy resistant epilepsy.This term is often used when patients have failed three or moreanti-epileptic drugs (AEDs). Refractory epilepsy also includesrefractory partial epilepsy and refractory generalized epilepsy(including idiopathic or symptomatic).

As used herein, the term “neuropathic pain” includes pain resulting fromchronic or debilitating conditions or disorders. The chronic ordebilitating conditions or disorders which can lead to neuropathic paininclude, but are not limited to, painful diabetic peripheral neuropathy,post-herpetic neuralgia, trigeminal neuralgia, post-stroke pain,multiple sclerosis-associated pain, neuropathies-associated pain such asin idiopathic or post-traumatic neuropathy and mononeuritis,HIV-associated neuropathic pain, cancer-associated neuropathic pain,carpal tunnel-associated neuropathic pain, spinal cord injury-associatedpain, complex regional pain syndrome, fibromyalgia-associatedneuropathic pain, lumbar and cervical pain, reflex sympathic dystrophy,phantom limb syndrome and other chronic and debilitatingcondition-associated pain syndromes.

As used herein, the term “migraine” shall mean a chronic, episodic anddebilitating clinical condition that is diagnosed by the presence ofmoderate to severe pulsating unilateral headaches lasting between 4 and72 h, which includes migraine without aura and migraine with aura. Asused herein, “migraine without aura” shall mean at least five attacksfulfilling the following criteria: (a) the headache attack lasts 4-72hours with the headache having at least two of the following features:unilateral location, pulsating quality, moderate or severe intensitywith direct influence on activities of daily living, and aggravation bywalking up stairs or similar routines: and (b) during the headache atleast one of the following occurs: nausea and/or vomiting, andphotophobia and phonophobia. As used herein, “migraine with aura” shallmean at least two attacks accompanied by at least 3 of the 4 followingfeatures: (a) one or more fully reversible aura symptoms: (b) at leastone aura symptom which develops gradually over more than four minutes ortwo or more symptoms which occur in succession; (c) no aura symptomwhich lasts more than 60 minutes; (d) a headache occurs prior to,simultaneously with or following the aura, with a free interval betweenaura and headache of less than about 60 minutes.

As used herein, the term “bipolar and related disorders” shall includebipolar disorder I (e.g. single manic episode, most recent episodehypomanic, most recent episode manic, most recent episode mixed, mostrecent episode depressed and most recent episode unspecified), bipolardisorder II, cyclothymic disorder and bipolar disorder not otherwisespecified (as these terms are defined by their diagnostic criteria, inthe Diagnostic and Statistical manual of Mental Disorders 4th Edition,Text Revision, American Psychiatric Association, 2000 (DSM-IV-TR) or inthe 5^(th) Edition, Text Revision, American Psychiatric Association,2013 (DSM-5™). Preferably, the bipolar disorder is characterized bydepressive and manic (or hypomanic) phases, wherein the phases cycle.Preferably, the bipolar disorder is bipolar disorder I or bipolardisorder II. As used herein “mania” shall include mania or a manic moodphase, regardless of underlying cause. As used herein, the term “bipolarmania” is intended to mean the mania associated with, characteristic ofor symptomatic of a bipolar disorder. Thus, methods of treating bipolarmania of the present invention are directed to methods which treat themania and/or manic phase of bipolar disorders. As used herein, the term“bipolar depression” is intended to mean the depression associated with,characteristic of or symptomatic of a bipolar disorder. Thus, methods oftreating bipolar depression of the present invention are directed tomethods which treat the depression and/or depressed phase of bipolardisorders. As used herein, unless otherwise noted the terms “cycling” or“bipolar cycling” shall refer to the alteration of mood betweendepressive and manic phases characteristic of bipolar disorders. Thus,the present invention includes methods for the stabilization of saidcycling, including, but not limited to, decreasing the frequency of thecycling and/or decreasing the magnitude of the manic and/or depressivephases.

Thus, in an embodiment, the pharmaceutical composition of the presentinvention may be used for mood stabilization, in particular moodstabilization for manic depression.

As used herein, the term “epileptogenesis” refers to the gradual processby which epilepsy develops. This process may occur following braininsults or a variety of conditions, including neurodegenerativediseases, traumatic brain injury, stroke, brain tumor, infections of thecentral nervous system, and status epilepticus; or it may occurfollowing gene mutations.

As used herein, the term “anxiety” refers in particular to generalizedanxiety disorder.

As used herein, the term “about” has its conventional meaning. Inparticular embodiments, when in relation to a numerical value, it may beinterpreted to mean the numerical value ±10%, or ±5%, or ±2%, or ±1%, or±0.5%, or ±0.1%. In other embodiments, the precise value is meant, i.e.by leaving out the word “about”.

“And/or” means that each one or both or all of the components orfeatures of a list are possible variants, especially two or more thereofin an alternative or cumulative way.

As used herein, the term “a”, “an”, “the” and similar terms used in thecontext of the present invention (especially in the context of theclaims) are to be construed to cover both the singular and plural unlessotherwise indicated herein or clearly contradicted by the context.

EXAMPLES

The following Examples are set forth to aid in the understanding of theinvention, and are not intended and should not be construed to limit inany way the invention set forth in the claims which follow thereafter.

A) Compounds of Formula (I-B)—Chemistry and In Vitro Testing

Several methods for preparing the compounds of Formula (I-B) of thisinvention are illustrated in the following Examples. Unless otherwisenoted, all starting materials were obtained from commercial suppliersand used without further purification.

Hereinafter, “aq.” means aqueous; “DCE” means 1,2-dichloroethane, “DCM”means dichloromethane; “DIPE” means diisopropylether; “DIPEA” meansN,N-diisopropylethylamine; “DMF” means N,N-dimethylformamide; “ES” meanselectrospray; “Et₃N” means triethylamine; “Et₂O” means diethyl ether;“EtOAc” means ethyl acetate; “h” means hours; “HPLC” means highperformance liquid chromatography; “HRMS” means high-resolution massspectra/spectrometry; “l” or “L” means liter; “LRMS” meanslow-resolution mass spectrometry/spectra; “MeOH” means methanol; “min”means minute(s); “mp” means melting point; “Pd(PPh₃)₄” meanstetrakis(triphenylphosphine)palladium(0); “RP” means reverse phase;“r.t.” means room temperature; “s” means seconds; “sat.” meanssaturated; “SFC” means supercritical fluid chromatography; “sol.” meanssolution; “THF” means tetrahydrofuran.

Microwave assisted reactions were performed in a single-mode reactor:Initiator™ Sixty EXP microwave reactor (Biotage AB), or in a multimodereactor: MicroSYNTH Labstation (Milestone, Inc.).

Thin layer chromatography (TLC) was carried out on silica gel 60 F254plates (Merck) using reagent grade solvents. Open column chromatographywas performed on silica gel, particle size 60 Å, mesh=230-400 (Merck)using standard techniques. Automated flash column chromatography wasperformed using ready-to-connect cartridges from Merck, on irregularsilica gel, particle size 15-40 μm (normal phase disposable flashcolumns) on a SPOT or LAFLASH system from Armen Instrument.

The absolute stereochemical configuration for some of the compounds wasdetermined using vibrational circular dichroism (VCD). They weremeasured on a Bruker Equinox 55 equipped with a PMA 37, in a KBr liquidcell using CD₂Cl₂ as solvent (PEM: 1350 cm-1, LIA: 1 mV, resolution: 4cm⁻¹). A description on the use of VCD for the determination of absoluteconfiguration can be found in Dyatkin A. B. et. al, Chirality,14:215-219 (2002).

Whenever the notation “RS” is indicated herein, it denotes that thecompound is a racemic mixture, unless otherwise indicated. Thestereochemical configuration for some compounds has been designated “R”or “S” when the mixture was separated; for some compounds, thestereochemical configuration has been designated as “*R” or “*S” whenthe absolute stereochemistry is undetermined although the compounditself has been isolated as a single stereoisomer and isenantiomerically pure. The enantiomeric excess of compounds reportedherein was determined by analysis of the racemic mixture bysupercritical fluid chromatography (SFC) followed by SFC comparison ofthe separated enantiomer(s).

Preparation of Intermediates

Description 1—Intermediate 1

Cyclopropylacetic acid ([CAS 5239-82-7], 50 g, 500 mmol) was dissolvedin CH₂Cl₂ (300 mL) then SOCl₂ (100 mL) was added. The reaction mixturewas stirred at 60° C. for 2 h and then the solvent was evaporated toyield intermediate 1 (53 g, 90%), which was used without furtherpurification.

Description 2—Intermediate 2

To a solution of 2,4-dichloro-3-iodopyridine ([CAS 343781-36-2], 290 g,1058 mmol) in DMF (1.7 L) was added methyl2,2-difluoro-2-(fluorosulfonyl)acetate ([CAS 680-15-9], 403 g, 2098mmol) and CuI (403 g, 2.13 mol), the reaction was then heated at 100° C.for 5 h.

The reaction was cooled and filtered. The filtrate was diluted with H₂Oand extracted with Et₂O and washed with a NH₃ solution. The organiclayer was dried (Na₂SO₄), filtered and concentrated in vacuo to yieldintermediate 2 (160 g), which was used without further purification.

Description 3—Intermediate 3

To a solution of NaH (60% in oil, 24 g, 600 mmol) in DMF (2 L) at 0° C.was added benzyl alcohol (35 g, 325 mmol), then the reaction was stirredfor 2 min. Intermediate 2 (160 mg, 741 mmol) was added in one portion,and stirred at 0° C. for 1 h. The reaction was diluted by the additionof H₂O and extracted with Et₂O. The organic layer was dried (Na₂SO₄),filtered and concentrated in vacuo. The residue was purified by columnchromatography over silica gel (eluent: petroleum ether/EtOAc=20/1). Thepure fractions were collected and the solvent was evaporated to yieldintermediate 3 (100 g, 38%).

Description 4—Intermediate 4

To a solution of intermediate 3 (100 g, 277 mmol) in 1,4-dioxane (1.5 L)was added NH₂NH₂ hydrate (85% solution in water, 300 g, 9.11 mol), thereaction was then heated in sealed tube at 160° C. for 2 h. The mixturewas concentrated in vacuo, dissolved in DCM, washed with NaHCO₃. Theorganic layer was dried (Na₂SO₄), filtered and concentrated in vacuo toyield intermediate 4 (90 g, 90%), which was used without furtherpurification.

Description 5—Intermediate 5

To a solution of intermediate 4 (90 g, 318 mmol) in CH₂Cl₂ (1.5 L) wasadded triethylamine (64.3 g, 636 mmol), the mixture was cooled to 0° C.,then a solution of intermediate 1 (53 g, 449 mmol) in CH₂Cl₂ was added.The solution was stirred at RT for 1 h. The reaction mixture was washedwith a sat. aq. sol. of NaHCO₃, and extracted with CH₂Cl₂. The organiclayer was dried (Na₂SO₄), filtered and concentrated in vacuo to yieldintermediate 5 (104.4 g, 90%).

The following intermediates were synthesized following a syntheticsequence analogous to that reported in Description 5 (D5).

Intermediate Acid chloride Conditions

propionyl chloride ([CAS 79-03-8]) Addition run at RT.

cyclobutaneacetyl chloride ([CAS 59543-38-3]) Conditions as in D5.

2-ethoxy-acetyl chloride ([CAS 14077-58-8]) Conditions as in D5.

butyryl chloride ([CAS 141-75-3]) Conditions as in D5.Description 6(a) Intermediate 9

To a solution of intermediate 5 (101 g, 277 mmol) in CH₃CN (1.2 L) wereadded phosphorus(V) oxychloride (84.7 g, 553 mmol) andN,N-diisopropylethylamine (71.3 g, 553 mmol). The reaction mixture wasstirred at 90° C. for 38 h. The reaction was then diluted with DCM andwashed with a Na₂CO₃ solution. The organic layer was dried (Na₂SO₄),filtered and concentrated in vacuo. The residue was purified by columnchromatography over silica gel (eluent: petroleum ether/EtOAc=4/1). Thepure fractions were collected and the solvent was evaporated to yieldintermediate 9 (31.39 g, 41%).

(b) Intermediate 10

The reaction was performed in 4 batches then combined for work up andpurification. To a solution of intermediate 6 (7 g, 20.6 mmol) in DCE(50 mL), was added N,N-diisopropylethylamine (3.96 mL, 22.69 mmol) andthen phosphorus oxychloride (2.12 mL, 22.69 mmol) and the reactionmixture was heated in a microwave at 150° C. for 5 min. Then DCM wasadded and the organic layer was washed with a sat. sol. of NaHCO₃, dried(Na₂SO₄) and concentrated in vacuo to afford the desired compound, whichwas purified by column chromatography (gradient elution: DCM 100% toMeOH.NH₃ 2% in DCM) to yield intermediate 10 (2.5 g, 49%).

The following intermediates were synthesized following a syntheticsequence analogous to that reported in Description 6(a) or (b).

Starting Intermediate material Conditions

Inter- mediate 7 Reaction performed as in (a) but in CH₃CN. After thereaction was com- plete, the reaction mixture was poured into ice/waterthen washed with NaHCO₃ sat. sol. And extracted with DCM, dried(Na₂SO₄), filtered and concentrated. Purification was performed in Spot(Si cartridge, eluent DCM/EtOAc up to 10-20%).

Inter- mediate 8 Reaction performed as in (b). Purification by flashcolumn chromatography (silica; EtOAc in DCM 0/100 to 40/60).

Inter- mediate 25 Reaction performed as in (a). Purification by flashcolumn chromatography (silica; MeOH in CH₂Cl₂, from 0/100 to 4/96).Description 7—Intermediate 13

(Ph₃P)₄Pd (2.096 g, 1.81 mmol) was added to a stirred solution ofintermediate 9 (10 g, 36.28 mmol) and4,4,5,5-tetramethyl-2-vinyl-1,3,2-dioxaborolane ([CAS 75927-49-0], 7.77mL, 43.53 mmol) in deoxygenated dioxane (30 mL) and a deoxygenatedNaHCO₃ saturated solution (30 mL) under nitrogen. The mixture wasstirred at 100° C. for 18 h. The mixture was diluted with EtOAc/waterand filtered through a pad of diatomaceous earth. The filtrate wastreated with brine and extracted with EtOAc. The organic layer wasseparated, dried (Na₂SO₄), filtered and the solvents evaporated invacuo. The crude product was purified by flash column chromatography(silica; EtOAc in CH₂Cl₂ 0/100 to 5/95). The desired fractions werecollected and concentrated in vacuo to yield intermediate 13 (6.08, 63%)as a yellow solid.

The following intermediates were synthesized following a syntheticsequence analogous to that reported in Description 7.

Starting Intermediate material Conditions

Inter- mediate 10 Reaction performed at 150° C. Purifi- cation by flashcolumn chroma- tography (silica; 7N solution of ammonia in methanol inDCM 0/100 to 1/9).

Inter- mediate 11 Extraction with DCM, purification by flash columnchromatography (silica; MeOH in DCM 4/96).

Inter- mediate 12 Purification by flash column chromatography (silica;EtOAc in DCM 0/100 to 10/90).

Inter- mediate 26 Reaction mixture performed at 150° C. in micro- wave.Purification by flash column chromatography (silica; EtOAc in DCM 0/100to 10/90).Description 8(a) Intermediate 17

Osmium tetraoxide (2.5% in t-BuOH, 10.103 mL, 0.781 mmol) and then,sodium periodate 12.53 g, 58.58 mmol) in water (48.5 mL) were added to asuspension of Intermediate 13 (6.08 g, 20.02 mmol) in dioxane (192 mL).The mixture was stirred at room temperature for 2 h.

The mixture was treated with water and EtOAc and it was filtered offthrough a pad of diatomaceous earth. The filtrate was extracted withEtOAc. The organic layer was separated, dried (Na₂SO₄), filtered and thesolvents evaporated in vacuo. The crude product was washed with Et₂O andit was filtered and dried to yield intermediate 17 (4.25 g, 79%) as abrown solid.

(b) Intermediate 18

A suspension of sodium periodate (5.04 g, 23.54 mmol) in distilled water(19 mL) was added to a stirred solution of osmium tetraoxide (2.5% int-BuOH, 4.06 mL, 0.31 mmol) and intermediate 14 (2.08 g, 7.85 mmol) indioxane (75 mL). The mixture was stirred at room temperature for 150min, and then the mixture was treated with sat NaHCO₃ and brine, andextracted with DCM. The organic layer was separated, dried (Na₂SO₄),filtered and concentrated in vacuo. The product was triturated with Et₂Oand filtered in vacuo, and finally put in desiccator at 50° C. for 18 h,to yield intermediate 18 (1.6 g, 80%) as a brown solid.

The following intermediates were synthesized following a syntheticsequence analogous to that reported in Description 8.

Starting Intermediate material Conditions

Inter- mediate 15 Procedure as in (a).

Inter- mediate 16 Procedure as in (a).

Inter- mediate 27 Procedure as in (a), order of addition: osmiumtetroxide was added to a stirred solution of intermediate 27 in1,4-dioxane, then a suspension of sodium periodate in water was addedand the reaction mixture was stirred for 2 h at RT. No filtrationthrough a pad of diatomaceous earth.Description 9(a) Intermediates 21a, 21b and 21c

Methylmagnesium bromide (1.4 M in THF, 12.40 mL, 17.37 mmol) was addeddropwise to a stirred suspension of intermediate 17 (4.25 g, 15.79 mmol)in THF (281.07 mL) at −20° C. under N₂ atmosphere. The mixture wasstirred at −20° C. for 45 minutes. The crude was treated with a sat.sol. of NH₄Cl and extracted with EtOAc. The organic layer was separated,dried (Na₂SO₄), filtered and concentrated in vacuo. The residue waspurified by flash column chromatography (silica; MeOH in DCM 0/100 to4/96). The desired fractions were collected and concentrated in vacuo toyield intermediate 21a (racemic mixture) (2.96 g, 66%). Intermediate 21a(1.82 g) was purified by chiral SFC: [Stationary phase: CHIRALPAK AD-H(5 μm 250×20 mm), Mobile phase: 80% CO₂, 20% EtOH] yielding 21b(R-enantiomer) (0.453 g, 10%) as a pale grey solid and intermediate 21c(S-enantiomer) (0.439 g, 10%).

(b) Intermediate 22

Methylmagnesium bromide (1.4 M in THF, 3.97 mL, 5.56 mmol) was addeddropwise to a stirred suspension of intermediate 18 (1.23 g, 5.06 mmol)in THF (90 mL) at −20° C. under N₂ atmosphere. The mixture was stirredat −20° C. for 45 minutes. The crude was treated with a sat. sol. ofNH₄Cl and extracted with EtOAc. The organic layer was separated, dried(Na₂SO₄), filtered and concentrated in vacuo. The residue was purifiedby flash column chromatography (silica; MeOH in DCM 0/100 to 4/96). Thedesired fractions were collected and concentrated in vacuo. The residuethus obtained was triturated with Et₂O to yield intermediate 22 (620 mg,35%) as a pale yellow solid. The following intermediates weresynthesized following a synthetic sequence analogous to that reported inDescription 9.

Starting Intermediate material Conditions

Inter- mediate 19 Procedure (b).

Inter- mediate 20 Procedure (b).

Inter- mediate 28 Procedure (b).

Intermediate 24a was further separated into Intermediate 24b andIntermediate 24c:

Preparation of the Final Compounds of Formula (I-B) Example 1

(a) Synthesis of Compounds 4-b, 6-b and 5-b

DIAD (2.07 mL, 10.52 mmol) was added dropwise to a stirred solution ofintermediate 21a (2 g, 7.01 mmol), 2,4-difluorophenol (1.00 mL, 10.52mmol) and triphenylphosphine (2.76 g, 10.52 mmol) in THF (74.18 mL) at0° C. and under nitrogen atmosphere. The mixture was stirred at 100° C.for 10 minutes under microwave irradiation. The mixture was diluted withEtOAc and washed with a sat. sol. of NaHCO₃. The organic layer wasseparated, dried (Na₂SO₄), filtered and concentrated in vacuo. Theresidue was purified by flash column chromatography (silica; MeOH in DCM0/100 to 97/3). The desired fractions were collected and concentrated invacuo. The residue was triturated with DIPE to give compound 4-b (1.46g, 52%) as a white solid, which was purified by chiral SFC [Stationaryphase: Chiralpak AD (5 μm 250*30 mm, Mobile phase: 85% CO₂, 15% iPrOH)],yielding compound 6-b (0.659 g, 24%) and compound 5-b (0.693 g, 25%).

(b) Alternative Synthesis of Compound 6-b

DIAD (31.06 μL, 0.16 mmol) was added dropwise to a stirred solution ofintermediate 21b (30 mg, 0.11 mmol), 2,4-difluorophenol (15.07 μL, 0.16mmol) and triphenylphosphine (41.38 mg, 0.16 mmol) in THF (1.11 mL) at0° C. and under nitrogen atmosphere. The mixture was stirred at 100° C.for 10 minutes under microwave irradiation. The mixture was diluted withEtOAc and washed with a sat. sol. of NaHCO₃. The organic layer wasseparated, dried (Na₂SO₄), filtered and concentrated in vacuo. Theresidue was purified by flash column chromatography (silica; MeOH in DCM0/100 to 97/3). The desired fractions were collected and concentrated invacuo. The residue was triturated with DIPE to give compound 6-b (40 mg,96%) as a white solid.

(c) Synthesis of Compound 6-b Hydrochloride Salt (.HCl)

DIAD (207.06 μL, 1.05 mmol) was added dropwise to a stirred solution ofintermediate 21b (200 mg, 0.70 mmol), 2,4-difluorophenol (100.45 μL,1.05 mmol) and triphenylphosphine (275.84 mg, 1.0516 mmol) in THF (4 mL)at 0° C. and under nitrogen atmosphere. The mixture was stirred at 100°C. for 15 minutes under microwave irradiation. The mixture was dilutedwith EtOAc and washed with a sat. sol. of NaHCO₃. The organic layer wasseparated, dried (Na₂SO₄), filtered and concentrated in vacuo. Theresidue was purified by RP HPLC (Stationary phase: C18 XBridge 30×100 mm5 μm, Mobile phase: Gradient from 60% 0.1% NH₄CO₃H/NH₄OH pH 9 solutionin Water, 40% CH₃CN to 43% 0.1% NH₄CO₃H/NH₄OH pH 9 solution in Water,57% CH₃CN), yielding a white solid residue that was dissolved in Et₂O (8mL) and 1,4-dioxane (0.5 mL). To the solution thus obtained HCl (4M indioxane, 200 μL) was added dropwise. The white solid precipitate wasfiltered, washed with Et₂O, dried (Na₂SO₄) and evaporated under vacuum.The white residue thus obtained was triturated with Et₂O to givecompound 6-b .HCl (110 mg, 36%) as a white solid.

The following compounds were synthesized following a synthetic sequenceanalogous to that reported in Example 1(b), starting from intermediate21b.

Co. No.

 9-b

10-b

11-b

12-b

13-b

14-b

Example 2

Synthesis of Compound 7-b

Procedure (a): DIAD (31.06 μL, 0.158 mmol) was added dropwise to astirred solution of intermediate 21b (30 mg, 0.105 mmol),3,5-difluorophenol (20.52 mg, 0.158 mmol) and triphenylphosphine (41.38mg, 0.158 mmol) in THF (1.113 mL) at 0° C. and under nitrogenatmosphere. The mixture was stirred at 100° C. for 10 minutes undermicrowave irradiation. The mixture was diluted with EtOAc and washedwith a sat. sol. of NaHCO₃. The organic layer was separated, dried(Na₂SO₄), filtered and concentrated in vacuo. The residue was purifiedby flash column chromatography (silica; MeOH in DCM 0/100 to 96/4). Thedesired fractions were collected and concentrated in vacuo. The residuewas triturated with DIPE to give compound 7-b (21 mg, 50%) as a whitesolid.

Procedure (b): Alternatively, compound 7 was also synthesized followinga synthetic sequence analogous to that reported in Example 1(b),starting from intermediate 21b.

Example 3

Synthesis of Compound 8-b

Procedure (a): DIAD (31.06 μL, 0.158 mmol) was added dropwise to astirred solution of intermediate 21b (30 mg, 0.105 mmol),3,4-difluorophenol (20.52 mg, 0.158 mmol) and triphenylphosphine (41.38mg, 0.158 mmol) in THF (1.11 mL) at 0° C. and under nitrogen atmosphere.The mixture was stirred at 100° C. for 10 minutes under microwaveirradiation. The mixture was diluted with EtOAc and washed with a sat.sol. of NaHCO₃. The organic layer was separated, dried (Na₂SO₄),filtered and concentrated in vacuo. The residue was purified by flashcolumn chromatography (silica; MeOH in DCM 0/100 to 96/4). The desiredfractions were collected and concentrated in vacuo. The residue wastriturated with DIPE to give compound 8-b (10.6 mg, 25%) as a whitesolid.

Procedure (b): Alternatively, compound 8-b was also synthesizedfollowing a synthetic sequence analogous to that reported in Example1(b), starting from intermediate 21b.

Example 4

Synthesis of Compound 15-b

Procedure (a): DIAD (155.3 μL, 0.789 mmol) was added dropwise to astirred solution of intermediate 21b (150 mg, 0.526 mmol),2,4,6-trifluorophenol (116.8 mg, 0.789 mol) and triphenylphosphine(206.88 mg, 0.789 mmol) in THF (5.56 mL) at 0° C. and under nitrogenatmosphere. The mixture was stirred at 100° C. for 10 minutes undermicrowave. The mixture was diluted with DCM and washed with a sat. sol.of NaHCO₃. The organic layer was separated, dried (Na₂SO₄), filtered andconcentrated in vacuo, then purified by flash column chromatography(silica; MeOH/NH₃ 7 N in DCM 0/100 to 90/10). The desired fractions werecollected and concentrated in vacuo. The was purified by RP HPLC(Stationary phase: C18 XBridge 30×100 mm 5 μm, Mobile phase: Gradientfrom 54% 0.1% NH₄CO₃H/NH₄OH pH 9 solution in Water, 46% CH₃CN to 64%0.1% NH₄CO₃H/NH₄OH pH 9 solution in Water, 36% CH₃CN) yielding acolourless oil that was crystallized upon standing (2 days). The solidwas triturated with heptane to give compound 15-b (129.8 mg, 59%) as awhite solid. Procedure (b): Alternatively, compound 15-b was alsosynthesized following a synthetic sequence analogous to that reported inExample 1(b), starting from intermediate 21b.

Example 5

Synthesis of Compounds 1-b, 2-b and 3-b

Compounds 1-b, 2-b and 3-b were synthesized following the proceduredescribed in Example 1(a). Thus, reaction of DIAD (500.05 μL, 2.54mmol), intermediate 21a (483 mg, 1.69 mmol), 4-fluorophenol (227.77 mg,2.03 mmol) and triphenylphosphine (666.14 mg, 2.54 mmol) in THF (17.91mL) as described in Example 1(a) yielded a residue that was purified byflash column chromatography (silica; EtOAc in DCM 0/100 to 90/10). Thedesired fractions were collected and concentrated in vacuo. Theresulting residue was triturated with DIPE to yield compound 1-b (320mg, 50%) as a white solid, which was purified by chiral SFC [Stationaryphase: Chiralpak AD (5 μm 250*30 mm, Mobile phase: 77% CO₂, 23% MeOH)],yielding compound 2-b (131 mg, 20%) and compound 3-b (129 mg, 20%) aswhite solids.

Example 6

Synthesis of Compounds 24-b, 26-b, and 27-b

Compounds 24-b, 26-b and 27-b were synthesized following the proceduredescribed in Example 1(a). Thus, reaction of DIAD (364.57 μL, 1.85mmol), intermediate 22 (320 mg, 1.23 mmol), 2,4-difluorophenol (176.86μL, 1.85 mmol) and triphenylphosphine (485.67 mg, 1.85 mmol) in THF(13.06 mL) as described in Example 1(a) yielded a residue that waspurified by flash column chromatography (silica; MeOH in DCM 0/100 to96/4). The desired fractions were collected and concentrated in vacuo toyield a colourless oil that crystallized with DIPE to give compound 24as a white solid, which was purified by RP HPLC (Stationary phase: C18XBridge 30×100 mm 5 μm; mobile phase: Gradient from 54% 0.1%NH₄CO₃H/NH₄OH pH 9 solution in Water, 46% CH₃CN to 64% 0.1%NH₄CO₃H/NH₄OH pH 9 solution in Water, 36% CH₃CN) yielding a colourlessoil that was crystallized upon trituration with heptane to give 240 mg(52%) of compound 24-b as a white solid, which was then purified bychiral SFC (Stationary phase: CHIRALPAK AD-H 5 μm 250×20 mm; mobilephase: 85% CO₂, 15% iPOH (0.3% iPrNH₂)), yielding compound 26-b (103 mg,22%) and compound 27-b (107 mg, 23%).

The following compounds were obtained following a synthetic sequencesimilar to that reported in Example 1(a).

The following compounds were synthesized following a synthetic sequenceas reported in Example 1(b), starting from the indicated intermediates.

Table A below lists additional compounds of Formula (I-B) which wereprepared by analogy to the above examples (Exp. no.).

TABLE A Example compounds according to Formula (I-B).

Co. Stereo- no. Exp no. R¹ Ar chem.  1-b E5^(#)

RS  2-b E5^(#)

*R  3-b E5^(#)

*S  4-b E1^(#)

RS  5-b E1^(#)

R  6-b      6-b •HCl E1(a) and (b)^(#) E1(c)^(#)

S  7-b E2^(#)

S  8-b E3^(#)

S  9-b E1(b)

S 10-b E1(b)

S 11-b E1(b)

S 12-b E1(b)

S 13-b E1(b)

S 14-b E1(b)

S 15-b E4^(#)

S 16-b E1(a)

RS 17-b E1(a)

*R 18-b E1(a)

*S 19-b E1(b)

RS 20-b E1(b)

*R 21-b E1(b)

*S 22-b E1(b)

*R 23-b E1(b)

*S 24-b E6^(#)

RS 25-b E1(a)

RS 26-b E6^(#)

*R 27-b E6^(#)

*S 28-b E1(a)

*R 29-b E1(a)

*S 30-b E1(b)

RS 31-b E1(b)

*R 32-b E1(b)

*S ^(#)indicates that the experimental procedure is described in theexamples.Analytical PartOptical Rotations

Optical rotations were measured on a Perkin-Elmer 341 polarimeter with asodium lamp and reported as follows: [α]° (λ, c g/100 ml, solvent, T°C.).

[α]_(λ) ^(T)=(100α)/(l×c): where l is the path length in dm and c is theconcentration in g/100 ml for a sample at a temperature T (° C.) and awavelength λ (in nm). If the wavelength of light used is 589 nm (thesodium D line), then the symbol D might be used instead. The sign of therotation (+ or −) should always be given. When using this equation theconcentration and solvent are always provided in parentheses after therotation. The rotation is reported using degrees and no units ofconcentration are given (it is assumed to be g/100 ml).

LCMS

For (LC)MS-characterization of the compounds of the present invention,the following methods were used.

General Procedure

The High Performance Liquid Chromatography (HPLC) measurement wasperformed using a LC pump, a diode-array (DAD) or a UV detector and acolumn as specified in the respective methods. If necessary, additionaldetectors were included (see table of methods below).

Flow from the column was brought to the Mass Spectrometer (MS) which wasconfigured with an atmospheric pressure ion source. It is within theknowledge of the skilled person to set the tune parameters (e.g.scanning range, dwell time . . . ) in order to obtain ions allowing theidentification of the compound's nominal monoisotopic molecular weight(MW). Data acquisition was performed with appropriate software.Compounds are described by their experimental retention times (R_(e))and ions. If not specified differently in the table of data, thereported molecular ion corresponds to the [M+H]⁺ (protonated molecule)and/or [M−H]⁻ (deprotonated molecule). In case the compound was notdirectly ionizable the type of adduct is specified (i.e. [M+NH₄]⁺,[M+HCOO]⁻, etc. . . . ). For molecules with multiple isotopic patterns(Br, Cl . . . ), the reported value is the one obtained for the lowestisotope mass. All results were obtained with experimental uncertaintiesthat are commonly associated with the method used. Hereinafter, “SQD”means Single Quadrupole Detector, “RT” room temperature, “BEH” bridgedethylsiloxane/silica hybrid, “HSS” High Strength Silica, “DAD” DiodeArray Detector.

TABLE B LCMS Method codes (Flow expressed in mL/min; column temperature(T) in ° C.; Run time in minutes). Flow Run LCMS Instrument ColumnMobile phase Gradient Col T time Method Waters: Agilent: A: 95% From 95%1 5 1 Acquity ® Eclipse Plus CH₃COONH₄ A to 5% A 50 UPLC ® - C18 RRHD6.5 mM + 5% in 4.6 min, DAD and (1.8 μm, CH₃CN, B: held for SQD 2.1 × 50mm) CH₃CN 0.4 min Waters: Waters: A: 95% From 95% 1 5 2 Acquity ® CSH ™C18 CH₃COONH₄ A to 5% A 50 UPLC ® - (1.7 μm, 6.5 mM + 5% in 4.6 min, DADand 2.1 × 50 mm) CH₃CN, B: held for SQD CH₃CN 0.4 min Waters: Waters:BEH A: 95% 84.2% A for 0.343 6.2 3 Acquity C18 (1.7 μm, CH₃COONH₄ 0.49min, to 40 UPLC ® - 2.1 × 100 mm) 7 mM/5% 10.5% A in DAD and CH₃CN, 2.18min, Quattro B: CH₃CN held for Micro ™ 1.94 min, back to 84.2% A in 0.73min, held for 0.73 min. Waters: Waters: A: 95% From 95% 1 9 4 Acquity ®CSH ™ C18 CH₃COONH₄ A to 5% A in 50 UPLC ® - (1.7 μm, 6.5 mM + 5% 7.8min, held DAD and 2.1 × 50 mm) CH₃CN, B: for 1.2 min SQD CH₃CNMelting Points

Values are peak values, and are obtained with experimental uncertaintiesthat are commonly associated with this analytical method.

Mettler FP 81HT/FP90 Apparatus

For a number of compounds, melting points were determined in opencapillary tubes on a FP 81HT/FP90 apparatus (Mettler-Toledo). Meltingpoints were measured with a temperature gradient of 1, 3, 5 or 10°C./minute. Maximum temperature was 300° C. The melting point was readfrom a digital display.

TABLE C Physico-chemical data for some compounds, retention time (R_(t))in min, [M + H]⁺ peak (protonated molecule), LCMS method and mp (meltingpoint in ° C.). (n.d. = not determined). Co. Mp R_(t) LCMS no. (° C.)(mm) [MH⁺] method Optical Rotation  1-b 156.3 2.32 380 1  2-b 176.9 2.93380 3 −58.5° (589 nm, c 0.53 w/v %, DMF, 20° C.)  3-b 177.3 2.93 380 3+59.4° (589 nm, c 0.52 w/v %, DMF, 20° C.)  4-b 121.7 2.41 398 1  5-b142 2.99 398.3 3 +95.7° (589 nm, c 0.69 w/v %, DMF, 20° C.)  6-b 142.42.99 398.2 3 −95.4° (589 nm, c 0.7 w/v %, DMF, 20° C.)  7-b 170.08 2.37398 2 −55.7° (589 nm, c 0.96 w/v %, DMF, 20° C.)  8-b n.d. 2.32 398 2n.d.  9-b n.d. 2.32 398 2 n.d. 10-b n.d. 2.25 398 2 n.d. 11-b n.d. 2.28398 2 n.d. 12-b n.d. 2.16 410 2 n.d. 13-b 144.1 2.68 410 2 n.d. 14-b161.7 2.51 394 2 n.d. 15-b 80.3 2.37 416 2 −167.0° (589 nm, c 0.55 w/v%, DMF, 20° C.) 16-b n.d. 2.50 412 2 n.d. 17-b n.d. 3.12 412 3 n.d. 18-bn.d. 3.12 412 3 n.d. 19-b n.d. 2.39 402 2 n.d. 20-b n.d. 2.3 402 2 n.d.21-b n.d. 3.36 402 n.d. 22-b n.d. 2.35 420 2 n.d. 23-b n.d. 2.35 420 2n.d. 24-b 135.7 2.05 372 2 n.d. 25-b 138.3 2.13 390 2 n.d. 26-b n.d.2.80 372 3 −83.9° (589 nm, c 0.52 w/v %, DMF, 25° C.) 27-b n.d. 2.80 3723 +92.1° (589 nm c 0.55 w/v %, DMF, 25° C.) 28-b n.d. 2.85 390 3 −129.2°(589 nm, c 0.5 w/v %, DMF, 25° C.) 29-b n.d. 2.85 390 3 +137.3° (589 nm,c 0.51 w/v %, DMF, 25° C.) 30-b 130.6 2.29 386 2 n.d. 31-b 127.85 2.29386 2 −67.5° (589 nm, c 0.83 w/v %, DMF, 20° C.) 32-b 127.69 2.29 386 2+89.5° (589 nm, c 0.83 w/v %, DMF, 20° C.)SFC-MSGeneral Procedure

The SFC measurement was performed using Analytical system from Bergerinstrument comprising a FCM-1200 dual pump fluid control module fordelivering carbon dioxide (CO2) and modifier, a CTC Analytics automaticliquid sampler, a TCM-20000 thermal control module for column heatingfrom room temperature to 80° C. An Agilent 1100 UV photodiode arraydetector equipped with a high-pressure flow cell standing up to 400 barswas used. Flow from the column was split to a MS spectrometer. The MSdetector was configured with an atmospheric pressure ionization source.The following ionization parameters for the Waters ZQ massspectrophotometer are: corona: 9 μa, source temp: 140° C., cone: 30 V,probe temp 450° C., extractor 3 V, desolvatation gas 400 L/hr, cone gas70 L/hr. Nitrogen was used as the nebulizer gas. Data acquisition wasperformed with a Waters-Micromass MassLynx-Openlynx data system.

Method 1:

In addition to the general procedure: The analytical chiral separationin SFC-MS was carried out on a CHIRALPAK AD DAICEL column (10 μm,4.6×250 mm) at 35° C. with a flow rate of 3.0 ml/min. The mobile phaseis 85% CO₂, 15% iPrOH (+0.3% iPrNH₂) hold 7 min in isocratic mode.

Method 2:

In addition to the general procedure: The analytical chiral separationin SFC-MS was carried out on a CHIRALPAK AD DAICEL column (10 μm,4.6×250 mm) at 35° C. with a flow rate of 3.0 ml/min. The mobile phaseis 75% CO2, 15% iPrOH (+0.3% iPrNH₂) hold 7 min in isocratic mode.

Method 3:

In addition to the general procedure: The analytical chiral separationin SFC-MS was carried out on a CHIRALPAK AD DAICEL column (10 μm,4.6×250 mm) at 35° C. with a flow rate of 3.0 ml/min. The mobile phaseis 80% CO2, 10% Methanol+10% iPrOH (+0.3% iPrNH₂) hold 7 min inisocratic mode.

TABLE D Analytical SFC data - R_(t) means retention time (in minutes),[M + H]⁺ means the protonated mass of the compound, method refers to themethod used for SFC/MS analysis of enantiomerically pure compounds. Themeasurement was compared against the mixture. Isomer Co. UV Area ElutionNr. R_(t) [M + H]⁺ % Method Order*  6-b 4.28 398 100 1 A  5-b 5.98 398100 1 B  2-b 2.13 380 100 2 A  3-b 2.97 380 100 2 B 17-b 2.46 412 100 3A 18-b 3.12 412 100 3 B 31-b 2.93 386 100 1 A 32-b 3.81 386 100 1 B *Ameans the first isomer that elutes. B means the second isomer thatelutes.Nuclear Magnetic Resonance (NMR)

For a number of compounds, ¹H NMR spectra were recorded either on aBruker DPX-400 or on a Bruker AV-500 spectrometer with standard pulsesequences, operating at 400 MHz and 500 MHz respectively. Chemicalshifts (δ) are reported in parts per million (ppm) downfield fromtetramethylsilane (TMS), which was used as internal standard.

Co. No. 6-b:

¹H NMR (400 MHz, CDCl₃) δ ppm 0.30-0.38 (m, 2H), 0.59-0.68 (m, 2H),1.14-1.22 (m, 1H), 1.72 (d, J=6.5 Hz, 3H), 3.02-3.14 (m, 2H), 5.84 (q,J=6.3 Hz, 1H), 6.67-6.73 (m, 1H), 6.80-6.89 (m, 2H), 7.30 (d, J=7.4 Hz,1H), 8.11 (d, J=7.4 Hz, 1H)

Co. No. 7-b:

¹H NMR (400 MHz, CDCl₃) δ ppm 0.30-0.39 (m, 2H), 0.59-0.68 (m, 2H),1.11-1.23 (m, 1H), 1.70 (d, J=6.5 Hz, 3H), 3.01-3.14 (m, 2H), 5.83 (q,J=6.2 Hz, 1H), 6.35-6.45 (m, 3H), 7.13 (d, J=7.2 Hz, 1H), 8.08 (d, J=7.4Hz, 1H)

Co. No. 8-b:

¹H NMR (400 MHz, CDCl₃) δ ppm 0.30-0.38 (m, 2H), 0.58-0.68 (m, 2H),1.11-1.22 (m, 1H), 1.69 (d, J=6.2 Hz, 3H), 3.01-3.13 (m, 2H), 5.79 (q,J=6.2 Hz, 1H), 6.53 (dtd, J=9.2, 3.1, 3.1, 1.7 Hz, 1H), 6.72 (ddd,J=11.6, 6.5, 3.1 Hz, 1H), 6.95-7.04 (m, 1H), 7.15 (d, J=7.4 Hz, 1H),8.07 (d, J=7.4 Hz, 1H)

Co. No. 15-b:

¹H NMR (500 MHz, CDCl₃) δ ppm 0.30-0.41 (m, 2H), 0.59-0.71 (m, 2H),1.16-1.25 (m, 1H), 1.70 (d, J=6.4 Hz, 3H), 3.05-3.16 (m, 2H), 5.80 (q,J=6.4 Hz, 1H), 6.62-6.70 (m, 2H), 7.45 (d, J=7.5 Hz, 1H), 8.16 (d, J=7.2Hz, 1H)

Co. No. 13-b:

¹H NMR (500 MHz, CDCl₃) δ ppm 0.27-0.39 (m, 2H), 0.58-0.67 (m, 2H),1.12-1.21 (m, 1H), 1.73 (d, J=6.4 Hz, 3H), 2.22 (s, 3H), 3.06 (qd,J=15.4, 6.6 Hz, 2H), 5.92 (q, J=6.4 Hz, 1H), 6.71 (d, J=8.4 Hz, 1H),6.89 (dd, J=8.4, 1.4 Hz, 1H), 7.18 (d, J=1.7 Hz, 1H), 7.32 (d, J=7.2 Hz,1H), 8.07 (d, J=7.2 Hz, 1H)

Co. No. 14-b:

¹H NMR (500 MHz, CDCl₃) δ ppm 0.28-0.39 (m, 2H), 0.57-0.69 (m, 2H),1.12-1.21 (m, 1H), 1.70 (d, J=6.6 Hz, 3H), 2.31 (s, 3H), 3.01-3.12 (m,2H), 5.79 (q, J=6.6 Hz, 1H), 6.55 (dd, J=9.0, 4.3 Hz, 1H), 6.69 (td,J=8.5, 3.0 Hz, 1H), 6.87 (dd, J=9.0, 2.9 Hz, 1H), 7.17 (d, J=7.5 Hz,1H), 8.06 (d, J=7.2 Hz, 1H)

Co. No. 20-b:

¹H NMR (500 MHz, CDCl₃) δ ppm 1.22 (t, J=7.1 Hz, 3H), 1.72 (d, J=6.4 Hz,3H), 3.58 (q, J=7.1 Hz, 2H), 5.03-5.10 (m, 2H), 5.84 (q, J=6.5 Hz, 1H),6.67-6.74 (m, 1H), 6.81-6.88 (m, 2H), 7.34 (d, J=7.2 Hz, 1H), 8.40 (d,J=7.5 Hz, 1H)

Co. No. 22-b:

¹H NMR (500 MHz, CDCl₃) δ ppm 1.23 (t, J=6.9 Hz, 3H), 1.70 (d, J=6.4 Hz,3H), 3.58 (q, J=7.0 Hz, 2H), 5.05-5.12 (m, 2H), 5.81 (q, J=6.6 Hz, 1H),6.62-6.70 (m, 2H), 7.48 (d, J=7.5 Hz, 1H), 8.45 (d, J=7.2 Hz, 1H)

Co. No. 31-b:

¹H NMR (400 MHz, CDCl₃) δ ppm 1.07 (t, J=7.40 Hz, 3H) 1.72 (d, J=6.24Hz, 3H) 1.92 (sxt, J=7.63 Hz, 2H) 2.98-3.14 (m, 2H) 5.84 (q, J=6.47 Hz,1H) 6.65-6.74 (m, 1H) 6.78-6.89 (m, 2H) 7.29 (d, J=7.40 Hz, 1H) 8.02 (d,J=7.40 Hz, 1H).

In Vitro Testing of Compounds of Formula (I-B)

The compounds of Formula (I-B) provided in the present invention arepositive allosteric modulators of mGluR2. These compounds appear topotentiate glutamate responses by binding to an allosteric site otherthan the glutamate binding site. The response of mGluR2 to aconcentration of glutamate is increased when compounds of Formula (I-B)are present. Compounds of Formula (I-B) are expected to have theireffect substantially at mGluR2 by virtue of their ability to enhance thefunction of the receptor. The effects of positive allosteric modulatorstested at mGluR2 using the [³⁵S]GTPγS binding assay method describedbelow and which is suitable for the identification of such compounds,and more particularly the compounds according to Formula (I-B), areshown in Table E.

[³⁵S]GTPγS Binding Assay

The [³⁵S]GTPγS binding assay is a functional membrane-based assay usedto study G-protein coupled receptor (GPCR) function wherebyincorporation of a non-hydrolysable form of GTP, [³⁵S]GTPγS (guanosine5′-triphosphate, labelled with gamma-emitting ³⁵S), is measured. TheG-protein α subunit catalyzes the exchange of guanosine 5′-diphosphate(GDP) by guanosine triphosphate (GTP) and on activation of the GPCR byan agonist, [³⁵S]GTPγS, becomes incorporated and cannot be cleaved tocontinue the exchange cycle (Harper (1998) Current Protocols inPharmacology 2.6.1-10, John Wiley & Sons, Inc.). The amount ofradioactive [³⁵S]GTPγS incorporation is a direct measure of the activityof the G-protein and hence the activity of the agonist can bedetermined. mGlu2 receptors are shown to be preferentially coupled toGαi-protein, a preferential coupling for this method, and hence it iswidely used to study receptor activation of mGlu2 receptors both inrecombinant cell lines and in tissues. Here we describe the use of the[³⁵S]GTPγS binding assay using membranes from cells transfected with thehuman mGlu2 receptor and adapted from Schaffhauser et al. (MolecularPharmacology, 2003, 4:798-810) for the detection of the positiveallosteric modulation (PAM) properties of the compounds of thisinvention.

Membrane Preparation

CHO-cells were cultured to pre-confluence and stimulated with 5 mMbutyrate for 24 h. Cells were then collected by scraping in PBS and cellsuspension was centrifuged (10 min at 4000 RPM in benchtop centrifuge).Supernatant was discarded and pellet gently resuspended in 50 mMTris-HCl, pH 7.4 by mixing with a vortex and pipetting up and down. Thesuspension was centrifuged at 16,000 RPM (Sorvall RC-5C plus rotorSS-34) for 10 minutes and the supernatant discarded. The pellet washomogenized in 5 mM Tris-HCl, pH 7.4 using an ultra-turrax homogenizerand centrifuged again (18,000 RPM, 20 min, 4° C.). The final pellet wasresuspended in 50 mM Tris-HCl, pH 7.4 and stored at −80° C. inappropriate aliquots before use. Protein concentration was determined bythe Bradford method (Bio-Rad, USA) with bovine serum albumin asstandard.

[³⁵S]GTPγS Binding Assay

Measurement of mGluR2 positive allosteric modulatory activity of testcompounds was performed as follows. Test compounds and glutamate werediluted in assay buffer containing 10 mM HEPES acid, 10 mM HEPES salt,pH 7.4, 100 mM NaCl, 3 mM MgCl₂ and 10 μM GDP. Human mGlu2receptor-containing membranes were thawed on ice and diluted in assaybuffer supplemented with 14 μg/ml saponin. Membranes were pre-incubatedwith compound alone or together with a predefined (˜EC₂₀) concentrationof glutamate (PAM assay) for 30 min at 30° C. After addition of[³⁵S]GTPγS (f.c. 0.1 nM), assay mixtures were shaken briefly and furtherincubated to allow [³⁵S]GTPγS incorporation on activation (30 minutes,30° C.). Final assay mixtures contained 7 μg of membrane protein in 10mM HEPES acid, 10 mM HEPES salt, pH 7.4, 100 mM NaCl, 3 mM MgCl₂, 10 μMGDP and 2 μg/ml saponin. Total reaction volume was 200 μl. Reactionswere terminated by rapid filtration through Unifilter-96 GF/B plates(Perkin Elmer, Massachusetts, USA) using a 96-well filtermate universalharvester. Filters were washed 6 times with ice-cold 10 mM NaH₂PO₄/10 mMNa₂HPO₄, pH 7.4. Filters were then air-dried, and 40 μl of liquidscintillation cocktail (Microscint-O) was added to each well.Membrane-bound radioactivity was counted in a Microplate Scintillationand Luminescence Counter from Perkin Elmer.

Data Analysis

The concentration-response curves of representative compounds of thepresent invention—obtained in the presence of EC₂₀ of mGluR2 agonistglutamate to determine positive allosteric modulation (PAM)—weregenerated using the Lexis software interface (developed at J&J). Datawere calculated as % of the control glutamate response, defined as themaximal response that is generated upon addition of glutamate alone.Sigmoid concentration-response curves plotting these percentages versusthe log concentration of the test compound were analyzed usingnon-linear regression analysis. The concentration producing half-maximaleffect is then calculated as EC₅₀.

The pEC₅₀ values below were calculated as the −log EC₅₀, when the EC₅₀is expressed in M. E_(max) is defined as relative maximal effect (i.e.maximal % effect relative to the control glutamate response).

Table E below shows the pharmacological data obtained for compounds ofFormula (I-B) and current pharmacological data obtained for compounds ofFormulae (I) and (I-A).

TABLE E Pharmacological data for compounds according to the invention.GTPγS- GTPγS- hmGluR2 hmGluR2 Co. No. PAM pEC₅₀ PAM E_(max)  1-b 6.59296  2-b 6.84 228  3-b 5.79 187  6-b 7.39 256  5-b 6.06 141  4-b 7.04329  7-b 7.31 292  8-b 7.04 244  9-b 7.3 260 10-b 7.47 218 11-b 8.25 23912-b 6.99 178 16-b 7.54 284 13-b 7.75 280 14-b 7.53 281 15-b 8.16 29319-b 6.71 297 25-b 6.9 233 24-b 6.42 193 17-b 7.73 317 18-b 6.24 21322-b 7.61 325 23-b 5.94 167 21-b 6.32 102 20-b 7.07 332 26-b 6.78 21427-b n.c. 51 30-b 6.9 227 28-b 7.19 234 29-b 5.85 77 31-b 7.05 251 32-b5.71 116  1 7.11 258  1a 6.95 286  2 7.82 290  2a 7.61 484  3 7.55 212 4 6.88 260  5 6.26 231  6 7.79 263  6a 7.68 261  7 8.45 263  8 6.73 360 9 6.9 462 10 7.21 357 11 6.94 310 12 8.36 261 13 6.9 278  1-a 6.78 314 2-a 6.84 340  3-a 6.88 231  4-a 6.6 269  5-a n.t.  6-a 6.34 255  7-a6.64 291  8-a 6.04 157  9-a 6.59 222 10-a 6.88 290 11-a 7.11 249 12-a7.03 242 13-a 6.67 212 14-a 6.92 259 15-a 7 253 16-a 7.12 223 17-a 6.54261 18-a nt. 19-a 6.71 240 20-a 6.91 243 21-a 6.25 207 22-a 6.05 25923-a 6.58 203 24-a 6.91 258 25-a 7.07 261 26-a 6.5 248 27-a 6.48 28428-a 6.96 297 29-a 6.97 317 30-a nt. 31-a nt. 32-a 6.66 347 33-a 6.58362 n.c. means that the pEC₅₀ could not be calculated n.t. means nottested pEC₅₀ values were not calculated in cases where theconcentration-response curve did not reach a plateau level.

All compounds were tested in presence of mGluR2 agonist glutamate at apredetermined EC₂₀ concentration, to determine positive allostericmodulation. pEC₅₀ values were calculated from a concentration-responseexperiment of at least 8 concentrations.

B) Anticonvulsant Studies with mGluR2 Compounds (Orthosteric Agonist andCompounds of Formulae (I)/(I-A)/(I-B)

General

Preparation of Test Compounds and Solutions

Test compounds were administered using an optimal fluid volume to bodyfluid ratio. Test compounds were administered to mice in a volume of0.01 mL/g of body weight (White H. S., et al., General Principles:Experimental selection, quantification, and evaluation of antiepilepticdrugs, in Antiepileptic Drugs, Fourth Edition, R. H. Levy, R. H.Mattson, and B. S. Meldrum, Editors. 1995, Raven Press, Ltd.: New York,pp. 99-110). For subcutaneous (s.c.) administration, the test compoundswere administered into a loose fold of skin along the back of the animalexcept compound 6-b, which was administered orally (p.o). For each ofthe tests performed on the test compounds (except on compound 6-b),final compound concentrations were administered as aqueous solution in20% Hp-β-CD. For compound 6-b, a 40% Hp-β-CD stock solution was firstprepared and utilized for formulating compound 6-b at the desiredconcentrations for testing via the oral route; final compoundconcentrations were administered as suspensions in 20% Hp-β-CD. A 20%Hp-β-CD solution was used for the vehicle groups.

For LY-404039, final compound concentrations were administered as asaline solution s.c.

For compound CAS 1092453-15-0, final compound concentrations wereadministered in 10% Hp-β-CD (+NaCl) vehicle following dissolution.

Final levetiracetam concentrations were administered in a 0.5%methylcellulose (MC) aqueous solution administered by intraperitoneal(i.p.) injection.

Critical Reagents

a) Vehicle Solutions

0.5% Methylcellulose (MC)

40% Hydroxypropyl-β-cyclodextrin (Hp-β-CD) stock solution

b) Miscellaneous Solutions

Tetracaine (0.5% solution w/v) was added dropwise from a plastic dropperbottle onto the eyes of all animals that would subsequently receiveelectrical stimulation via corneal electrodes.

Animals and Animal Husbandry

Adult male CF No 1 albino mice (26-35 g) were obtained from CharlesRiver, Portage, Mich. The animals were maintained on an adequate diet(Prolab RMH 3000) and allowed free access to food and water, exceptduring the short time they were removed from their cage for testing.Animals newly received in the laboratory were allowed sufficient time tocorrect for possible food and water restriction incurred during transitbefore being employed in testing. All mice were housed in plastic cagesin specially constructed rooms with controlled humidity, exchange of airand controlled lighting (12 hours on-12 hours off). The animals werehoused, fed, and handled in a manner consistent with the recommendationsin the National Council Publication, “Guide for the Care and Use ofLaboratory Animals”.

Minimal Motor Impairment (MMI)

Acute MMI was assessed by a combination of direct observations of theanimal for overt symptoms of the animal's neurological or muscularfunction. In mice, the rotarod procedure was used to disclose minimalmuscular or neurological impairment. When a mouse is placed on a rodthat rotates at a speed of 6 rpm, the animal can maintain itsequilibrium for long periods of time. The animal was considered toxic ifit fell off this rotating rod three times during a 1 min period.

Determination of Median Effective and Toxic Doses (ED₅₀ and TD₅₀)

In the determination of an ED₅₀ or TD₅₀ for each test compound, thefirst dose administered is usually the same dose as that used in asuccessful TPE determination.

If the initial dose employed was effective or toxic in more than 50% ofanimals, the next dose would be one-half that of the initial dose; ifthe initial dose was effective or toxic in less than 50% of animals, thefollowing dose would be twice that of the initial dose. Third and fourthdoses were selected to produce an evenly spaced dose response line.There should be a minimum of 4 points either including or lying between0 and 100%.

TPE Determination

Groups of generally four animals each were administered test compoundsand each group was tested at one of five time points: 0.25, 0.5, 1, 2,or 4 h post-treatment (White et al. 1995). TPE was determined using the6 Hz (32 mA) assay. The time (0.25, 0.5, 1, 2, or 4 h post-treatment) atwhich maximal protection was observed was considered the Time of PeakEffect (TPE).

At the TPE determined for this study, or determined previously,compounds were tested in the 6 Hz assay (32 and/or 44 mA), acrossseveral doses and comprising doses that elicited little or no protectionto full protection.

An ED₅₀ and 95% confidence interval (C.I.) were calculated using Probitanalysis on a computer program provided in the laboratory (Finney“Probit Analysis” 34d Ed 1971, London: Cambridge University Press).

Serum Collection for pK/pD Analysis

In various tests, animals were sacrificed following testing, and trunkblood and/or brain tissue (whole brains) was collected forquantification of drug levels. Immediately after testing, animals weredecapitated and trunk blood was collected into a BD Vacutainer® tubecontaining K2EDTA and chilled on ice until centrifugation. Followingcentrifugation (13000-18000 rpm, 5-7 min), the plasma was removed andtransferred to a labeled microcentrifuge tube and stored at −80° C. Forbrain tissue collection, brains were removed immediately followingdecapitation and flash frozen. The frozen sample was placed in a labeledcentrifuge tube and stored at −80° C.

6 Hz Psychomotor Seizure Test in Mice

The 6 Hz seizure test is used as a model of pharmacoresistant limbicseizures. The 6 Hz seizure displays a resistance to phenytoin,carbamazepine, lamotrigine, and topiramate (Barton et al.“Pharmacological characterization of the 6 Hz psychomotor seizure modelof partial epilepsy” Epilepsy Research 2001, Vol. 47, pp. 217-222).

Method for 6 Hz Psychomotor Seizure Test

Focal seizures were induced in mice via corneal stimulation (6 Hz, 0.2msec rectangular pulse, 3 sec duration; Barton et al. 2001). Mice weretested at either 32 mA or 44 mA. Prior to stimulation, drops of 0.5%tetracaine were applied to each eye. The seizures that arise fromcorneal stimulation in this assay are characterized by a minimal clonicphase followed by stereotyped automatistic behaviors including stun,forelimb clonus, twitching of the vibrissae, and Straub-tail. Animalsnot displaying these behaviors were considered protected.

Example 1—Studies with Compounds 1 and 2

1.1. Combination Study with Co. No. 1, Co. No. 2 and Levetiracetam

First, each compound was tested individually at a dose that displayedminimal activity in the 6 Hz 44 mA test at each compound's TPE. When themGluR2 PAM compounds and levetiracetam were administered in combination(same dose and time-point as individual tests) nearly completeprotection was observed in the 6 Hz 44 mA test (Table 2). In addition torecording the efficacy and toxicity data for these compounds alone or incombination, both plasma and brain samples were collected from each ofthe groups for pharmacokinetic/pharmacokinetic analysis. Nopharmacokinetic interaction was observed based on compound levels in theplasma and brain samples (data not shown). In summary, compounds 1 and 2displayed positive pharmacodynamic interaction with levetiracetam in the6 Hz model that does not appear to be due to pharmacokineticinteraction, and without increasing motor impairment (Tables 2, 2a, 2b).The effect of 1 dose of Compound 2 was also tested on the dose-responseof LEV. As shown in Table 3, there was a ˜200-fold shift in the ED₅₀ ofLEV compared to when LEV was tested alone. LEV seemed to increase thepotency of Co No. 2 slightly (Table 3).

1.2. Isobolographic Analysis of Interactions Between Co. No. 1 andLevetiracetam in the 6 Hz Seizure Model

Isobolographic studies were conducted for the combined administration ofCo. No. 1 with LEV in the 6 Hz (44 mA) assay. Studies were conductedaccording to previously described methods (Madsen et al. 2011). InitialED₅₀ values were determined for both Co. No. 1 and LEV and used tocalculate theoretical ED₅₀ (±standard error of the mean, SEM) values forthree fixed dose ratio combinations (LEV:Co. No. 1): 1:3, 1:1, and 3:1.Doses used were proportional to calculated ED₅₀ values. For example, thedose ratio used for the 1:1 paradigm was based on 0.5×ED₅₀ for LEV and0.5×ED₅₀ for Co. No. 1. Similarly, the 1:3 paradigm used 0.25×ED₅₀ forLEV and 0.75×ED₅₀ for Co. No. 1. The 3:1 dose ratio used 0.75×ED₅₀ LEVand 0.25×ED₅₀ for Co. No. 1. Experimental treatment doses (see Table 4)were based on theoretical values and adjusted according to observedeffects. Experimentally determined ED₅₀ (±SEM) values for each fixeddose-ratio combination were compared to the theoretical values (t-test)for statistical purposes. The dose ratio was determined to besupra-additive (synergistic) if the experimentally-determined ED₅₀ valuewas significantly lower than the theoretical ED₅₀. Subsequently, theexperimental combined doses were determined for the same paradigms inthe 6 Hz seizure test (Table 4 below). The isobolographic study withcompound 1 and levetiracetam in the 6 Hz model demonstrates asignificant synergistic pharmacodynamic interaction at all dose ratiosevaluated and corresponds closely with compound 1 plasma levels.Furthermore, no motor impairment was observed at any of the dose ratiosevaluated suggesting that the synergistic pharmacodynamics interactiondoes not produce increased motor toxicity.

1.3. Mouse Corneal Kindling Model and Studies with Compound 1

Mice were kindled electrically with 3 second, 3 mA, 60 Hz stimulus,twice daily using corneal electrodes until a criterion of 5 consecutiveStage 5 seizures as defined by Racine (Racine “Modification of seizureactivity by electrical stimulation” II. motor seizure” ElectroencephClin Neurophysiol 1972, 32, pp. 281-294). After the mice reached astable kindled state, the test compound or vehicle was administered and,at the previously determined TPE, each animal was given the electricalstimulus indicated above. Following stimulation, the animals wereobserved for the presence or absence of the seizure activity scored onthe Racine scale (0-5) with 5 representing the highest stage rearing andfalling. One dose of LEV and two doses of Co. No. 1 were testedindividually and in combination against corneal kindled seizures.Combination of compound 1 with levetiracetam in this model suggests apositive pharmacodynamics interaction (Table 5 below).

A summary of the data for the compounds tested alone is presented inTable 1 and additional results of studies performed according to example1 are listed in Tables 2-5 below.

TABLE 1 Summary of the acute anticonvulsant data in the 6 Hz model at 32mA and 44 mA for the mGluR2 PAM compounds 1, 2, 11, 2-a, 25-a, 6-b andLY-404039 following s.c. administration (except compound 6-b, which wastested p.o.). TPE means time of peak effect, CI means confidenceinterval, s.c. means subcutaneous, p.o. means orally, n.t. means nottested. TPE was determined in 32 mA 6 Hz test. Effects are generallyobserved at doses that do not produce impairment in rotarod test. Forcompounds 11 and 2-a the individual values of repeat experiments areprovided. For compound 25a, both 0.25 and 1 h time points were used for6 Hz (44 mA) studies. Seizure score (dose) TPE ED₅₀ (95% CI) mg/kg, s.c.Corneal Co. No. (h) 32 mA 44 mA kindling 11 0.5 4.77 (3.54-6.76) 31.5(15.1-47.3) 2.8 9.41 (1.53-15.1) (100 mg/kg)  2 0.25 3.83 (1.62-6.71)5.89 (3.89-8.45) 3.4 (40 mg/kg)  1 0.5 2.8 (1.3-4.3) 10.2 (3.1-12.4) 3.7(20 mg/kg) 25-a 1 7.7 (2.3-18.4) 1 hr TPE: 25.9 n.t. (15.5-33.7) 0.25 hrTPE: 29.1 (21.6-39.6)  2-a 0.5 44.7 (23.4-80.5) 50% protection at 4.420.8 (10.0-31.7) 100 mg/kg (100 mg/kg) 12.2 (8.4-17.4) 21 (17.9-27.4) 6-b 0.5 7.2 (4.2-11.8) 16.1 (13.0-20.1) n.t. LY-404039 0.5 10.2(3.62-12.4) n.t. 3.1 (100 mg/kg)

TABLE 1a Summary of the 6 Hz 32 mA TPE determination for Co. No. 1 DoseTime 6 Hz Motor (mg/kg, s.c.) (h) 32 mA impairment 10 4 0/4 0/0 2 0/40/0 1 3/4 0/0 0.5 4/4 0/0 0.25 4/4 0/0 5 0.5  8/12 0/0 0.25  8/12 0/02.5 0.5 5/8 0/0 0.25 1/8 0/0 (number of mice protected in 6 Hz or toxicon rotarod/number tested)

TABLE 1b Dose-response studies for Co. No. 1. The TPE for Co. No. 1 waspreviously determined to be 0.5 h (results shown above in table 1a).Several doses of Co. No. 1 were administered at this TPE and tested inthe 6 Hz assay, using both 32 and 44 mA stimulus intensities. Dose#rotarod motor Test (mg/kg, s.c.) #protected/#tested impairment/#tested6 Hz 32 mA 20 8/8 1/8 10 7/8 0/8 5  8/12  0/12 2.5  7/16  0/16 0.5 1/80/8 ED₅₀ (95% CI): 2.8 mg/kg (1.3 to 4.3) 6 Hz 44 mA 20 8/8 1/8 15 7/80/8 10 4/8 0/8 2.5 0/8 0/8 ED₅₀ (95% CI): 10.2 mg/kg (3.1 to 12.4)

TABLE 2 Summary of interaction of Co. No. 1 and Co. No. 2 withLevetiracetam (LEV) in the mouse 6 Hz, 44 mA seizure model. Results arelisted as number of mice exhibiting full protection/total number of micetested in each dosing group (at the specified test compound orcombination dosage levels). Time # protected/ # motortox/ Dose (h) #tested # tested LEV 10 mg/kg i.p. 1 1/6 0/6 Co. No. 2 + 3 mg/kg s.c.0.25 5/6 0/6 LEV Co. No. 2 3 mg/kg s.c. 0.25 1/6 0/6 LEV 10 mg/kg i.p. 11/8 0/8 Co. No. 1 + 2.5 mg/kg s.c. 0.5 6/8 0/8 LEV Co. No. 1 2.5 mg/kgs.c. 0.5 0/8 0/8

TABLE 2a Plasma and brain levels Co. No. 1. in combination study withLevetiracetam (LEV). BQL means below quantifiable limit. Plasma Plasma 6Hz Co. No. 1 LEV (ng/ml) Co. No. 1 (ng/ml) Protection 6 Hz 44 mA 10mg/kg 9350 2.5 mg/kg BQL Yes 8580 244 No 10900 314 Yes 10300 382 Yes9780 416 Yes 9780 377 Yes 13700 2260* No 10100 607 Yes Mean Plasma 10311 657.1 6/8 Level (390) Mean Plasma 1/8 8254 0/8 438 Levels (non-combination) Mean plasma level shown in parenthesis ( ) is calculatedwith a statistical outlier* removed

TABLE 2b Plasma and brain levels Co. No. 2 in combination study withLevetiracetam (LEV). AQL means above quantifiable limit. Plasma BrainPlasma Brain 6 Hz Co. No. 2 LEV (ng/ml) (ng/ml) Co. No. 2 (ng/ml)(ng/ml) Protection 6 Hz 44 mA 10 mg/kg 6450 6290 3 mg/kg 1830 1540 Yes8200 7990  386 1020 Yes 3540 4760 4700 1310 Yes 3850 NA  467 NA No 71506380 AQL (>500) 1120 Yes 3890 3960 2080 1140 Yes Mean 5513 5876 18931226 5/6 Plasma/ Brain Levels Mean 1/6 8750 5773 1/6 1295 1113 Plasma/Brain Levels (non- combination) NA - sample not available for analysis

TABLE 3 6 Hz seizure (44 mA) model ED₅₀ determinations for No. Co. 2 andlevetiracetam (LEV) alone and in combination. LEV at a dose of 10 mg/kgincreased the potency of the No. Co. 2 (~5-fold shift in ED₅₀). Co. No.2 at a dose of 3 mg/kg increased both the efficacy (to 100% protection)and potency of LEV (~200-fold shift in ED₅₀). FIG. 1 shows thedose-response for the 6 Hz 44 mA ED₅₀ determinations for the Co. No. 2and LEV alone and in combination. ED₅₀ (95% CI) Maximum Effect Treatmentmg/kg (% Protection) Co. No. 2 alone 6.97 100% (5.44-8.30) Co. No. 2 +LEV 1.35 100% (10 mg/kg) (0.8-1.9) LEV alone ~200  75% LEV + Co. No. 21.0 100% (3 mg/kg) (0.23-2.24)

TABLE 4 Results of Co. No. 1 and Levetiracetam in Isobolographic Study.6 Hz LEV Co. No. 1 combined Rotarod (44 mA) (mg/kg (mg/kg dose#motortox/ #protected/ Group i.p.) f s.c.) f (mg/kg) # tested #tested1:1 181 0.5 5.1 0.5 93.1 0/8 8/8 paradigm 90.5 2.6 46.6 0/8 6/8 45.3 1.323.3 0/8 3/8 22.6 0.6 11.6 0/8 3/8 ED₅₀ (95% CI; mg/kg): 22.2 (8.4-35.7)1:3 45.3 .25 3.8 .75 14.2 0/8 8/8 paradigm 22.6 1.9 7.1 0/8 4/8 11.3 1.03.6 0/8 2/8 ED₅₀ (95% CI; mg/kg): 5.9 (3.5-8.7) 3:1 271.5 .75 2.6 .25204.3 0/8 8/8 paradigm 135.8 1.3 102.2 0/8 3/8 67.9 0.6 51.1 0/8 3/833.9 0.3 25.5 0/8 0/8 ED₅₀ (95% CI; mg/kg): 86.3 (56.8-131.4)

The isobolographic analysis (FIG. 2) demonstrates that the combinationof Co. No. 1 and levetiracetam results in a significantly positivesynergistic effect.

TABLE 5 Results of Co. No. 1 and Levetiracetam combination study in thecorneal kindling model in mice. Mean # protected/ % Seizure Compound(s)# tested Protected Score Vehicle 0/10  0% 4.7 (20% HPBCD@30′, s.c.; 0.5%MC@60′, i.p.) LEV 3 mg/kg 5/13 38% 3.3 Co. No. 1 30 mg/kg 3/12 25% 4.0LEV 3 mg/kg & Co. No. 1 10/10  100%  0.6 30 mg/kg Co. No. 1 20 mg/kg5/16 31% 3.7 LEV 3 mg/kg & Co. No. 1 7/10 70% 1.9 20 mg/kg RacineSeizure Score 0 to 5 0 = no seizure activity 5 = maximal seizureactivity

Example 2—Studies with Compounds 25-a and 2-a

2.1. Combination Study with Co. No. 25-a and Levetiracetam

Independent dose-response studies were performed in the 6 Hz 44 mA testfor both compounds to determine ED₅₀ values at the TPE of 1 h i.p. forlevetiracetam and 1 h s.c. for Co. No. 25-a. The ED₅₀ value for Co. No.25-a was 25.9 mg/kg and for levetiracetam the value was estimated to beapproximately 345 mg/kg. The dose-response for levetiracetam wasrepeated with co-administration of 10 mg/kg Co. No. 25-a (a dose of Co.No. 25-a that alone did not protect in the 6 Hz 44 mA model). Theco-administration of 10 mg/kg Co. No. 25-a produced an ED₅₀ in thelevetiracetam dose-response of 4.9 mg/kg (˜70-fold lower compared withlevetiracetam alone) and importantly yielded full protection in the 6 Hz44 mA seizure model. These results are suggestive of a positivepharmacodynamic interaction in the 6 Hz seizure model between Co. No.25-a and levetiracetam.

TABLE 6 Time-to-Peak Effect Determination for Co. No. 25-a in the 6 Hz(32 mA) Assay. Two doses were used in this study, 10 and 20 mg/kg,across several time points (0.25-4 h). The compound showed the greatestdegree of protection in the 6 Hz assay between 0.25 and 1 h, which wasmore evident at 20 mg/kg. Plasma levels of the compound generallycorresponded to behavioural seizure protection. A TPE of 0.25 h was usedfor 6 Hz (32 mA) studies whereas both the 0.25 and 1 h time points wereused for 6 Hz (44 mA) studies. # rotarod motor Co. No. 25-a mean DoseTime # protected/ impairment/ plasma levels (mg/kg, s.c.) (h) # tested #tested (ng/mL) 10 0.25 2/4 0/4 10,983 (2,477) 0.5 1/4 0/4 3,330 1 1/41/4 700 2 0/4 0/4 256 4 0/4 0/4 40 20 0.25 4/4 0/4 4,095 0.5 3/4 1/42,800 1 4/4 1/4 1,765 2 1/4 0/4 618 4 1/4 1/4 28 Mean plasma level shownin parenthesis ( ) is calculated with a statistical outlier removed.s.c. means subcutaneous

TABLE 7 Dose-Response Studies for Co. No. 25-a in the 6 Hz Assay (32mA^(a) and 44 mA^(b)) Dose # rotarod motor Co. No. 25-a mean (mg/kg, #protected/ impairment/ plasma levels Test s.c.) # tested # tested(ng/mL) 6 Hz 20 8/8 0/8 5,570 32 mA 15 3/8 0/8 1,201 10 4/8 0/8 6,113 54/8 0/8 2,558 1 1/8 0/8 466 ED₅₀ (95% CI): 7.7 mg/kg (2.3 to 18.4) 6 Hz40 7/8 0/8 6,263 44 mA 30 3/8 0/8 7,220 20 2/8 0/8 3,368 10 0/8 0/84,345 (1,526) 5 0/8 1/8 1,428 ED₅₀ (95% CI): 29.1 mg/kg (21.6 to 39.6)^(a)The time-to-peak effect in the 6 Hz 32 mA assay for Co. No. 25-a wasdetermined to be 0.25 h (see Table 1). ^(b)The time-to-peak effect inthe 6 Hz 44 mA assay for Co. No. 25-a was similar for 0.25 h and 1 h;results for 1 h confirmed the ED₅₀ (95% CI) 25.9 (15.5-33.7) (see Table1 and 6). Mean plasma level shown in parenthesis ( ) is calculated witha statistical outlier removed. CI means confidence interval

TABLE 8 Combination Studies for Co. No. 25-a with Levetiracetam (LEV) inthe 6 Hz (44 mA) Assay. Dose # protected/ # rotarod motor Drug (mg/kg,s.c.) # tested impairment/# tested LEV 200 2/8 0/8 400 4/9 0/9 800 10/12 0/12 ED₅₀ (95% CI): 345.4 mg/kg (211.0 to 485.3) LEV + Co. No. 25-a 2008/8 1/8 10 mg/kg 100 7/8 2/8 50 5/8 1/8 10 4/8 0/8 1 4/8 1/8 ED₅₀ (95%CI): 4.9 (0.0-14.2) Co. No. 25-a (s.c.) 10 mg/kg tested in combinationwith LEV (i.p.) - Co. No. 25-a 10 mg/kg, not active when administeredalone.2.2. Combination Study with Co. No. 2-a and Levetiracetam

Dose-response studies were performed in the 6 Hz 32 mA and 44 mA tests(table 9 below) and in the combination test with levetiracetam (effectof Co. No. 2-a on the dose-response of LEV in tables 10a and effect ofLEV on the dose-response of Co. No. 2-a in table 10b below) in the samemanner as described for the studies with Co. No. 25-a and levetiracetamabove.

TABLE 9 Dose-Response Studies for Co. No. 2-a in the 6 Hz Assay (32 mAand 44 mA; 0.5 h TPE). A time-to-peak effect of 0.5 h was determined inthe 32 mA 6 Hz test (s.c.) and used for 6 Hz (32 mA and 44 mA) studies.Dose # protected/ # rotarod motor Test (mg/kg, s.c.) # testedimpairment/# tested 6 Hz 40 8/8 2/8 32 mA 20 6/8 3/8 10 4/8 0/8 5 0/80/8 2.5 0/8 1/8 ED₅₀ (95% CI): 12.2 mg/kg (8.4 to 17.4) 6 Hz 40 8/8 4/844 mA 20 3/8 0/8 3/8 0/8 15 2/8 1/8 10 0/8 1/8 0/8 0/8 ED₅₀ (95% CI):21.0 mg/kg (17.9 to 27.4) TD₅₀: >40 mg/kg^(a) ^(a)40 mg/kg - 6 out of 16total (32 mA and 44 mA combined) with impairment. Dose selected forcombination studies with LEV in 6 Hz (44 mA): Co. No. 2-a 10 mg/kg.

TABLE 10a Combination Studies for Co. No. 2-a with Levetiracetam (LEV)in the 6 Hz (44 mA) Assay. Combination of 10 mg/kg Co. No. 2-a withvarying doses of levetiracetam. Dose # protected/ # rotarod motor Drug(mg/kg) # tested impairment/# tested LEV 200 2/8 0/8 400 4/9 0/9 80010/12  0/12 LEV ED₅₀ (95% CI): 345.4 mg/kg (211.0 to 485.3) LEV + Co.No. 2-a 200 6/8 1/8 10 mg/kg^(a) 100 6/8 0/8 50 6/8 0/8 25 8/8 0/8 12.55/8 0/8 6.25 4/8 0/8 3.125 3/8 1/8 1.5625 0/8 0/8 LEV ED₅₀ (95% CI): 9.6mg/kg (1.7-21.9) ^(a)Co. No. 2-a (s.c.) 10 mg/kg tested in combinationwith LEV (i.p.); Co. No. 2-a 10 mg/kg, not active when administeredalone. Additional LEV (low-dose) control groups were tested at 25 and6.25 mg/kg (1/8 and 0/6 protected, respectively). Vehicle-treated mice(0.5% methylcellulose i.p. (1 h)/20% HPBCD s.c. (0.5 h)) showed noprotection (0/8 protected).

TABLE 10b Combination Studies for Co. No. 2-a with Levetiracetam (LEV)in the 6 Hz (44 mA) Assay. Combination of 350 mg/kg levetiracetam withvarying doses of Co. No. 2-a. Dose # protected/ # rotarod motor Drug(mg/kg) # tested impairment/# tested LEV (alone)^(a) 350 3/8 0/8 LEV 350mg/kg + 20 8/8 2/8 Co. No. 2-a^(b) 10 7/8 1/8 5 7/8 1/8 2.5 5/8 0/8 1.254/8 0/8 previous Co. No. 2-a ED₅₀ (95% CI): 21.0 mg/kg (17.9 to 27.4)LEV combination Co. No. 2-a ED₅₀ (95% CI): 1.5 mg/kg (0.1-2.7) ~14-foldshift in potency ^(a)LEV ED₅₀ (presented separately) previouslydetermined in 6 Hz (44 mA): 345 mg/kg. ^(b)Co. No. 2-a (s.c.) 10 mg/kgtested in combination with LEV (i.p.); Co. No. 2-a 10 mg/kg, not activewhen administered alone. Additional LEV (low-dose) control groups weretested at 25 and 6.25 mg/kg (1/8 and 0/6 protected, respectively).Vehicle-treated mice (0.5% methylcellulose i.p. (1 h)/20% HPBCD s.c.(0.5 h) showed no protection (0/8 protected).

At a dose of 10 mg/kg s.c., Co. No. 2-a increases the potency of LEV,leading to an approximate 35-fold shift in the ED₅₀. This suggests apositive pharmacodynamic relationship (Table 10a). At a dose of 350mg/kg i.p., LEV increases the potency of Co. No. 2-a, leading to anapproximate 14-fold shift in the ED₅₀. This suggests a positivepharmacodynamics relationship (Table 10b).

Example 3—Studies with Compound 6-B

3.1. Combination Study with Co. No. 6-b and Levetiracetam

Independent dose-response studies were performed in the 6 Hz 44 mA testfor both compounds to determine ED₅₀ values at the TPE of 1 h i.p. forlevetiracetam and 0.5 h p.o. for Co. No. 6-b. The ED₅₀ value for Co. No.6-b was 16.1 mg/kg and for levetiracetam the value was estimated to beapproximately 345 mg/kg. The dose-response for levetiracetam wasrepeated with co-administration of 10 mg/kg Co. No. 6-b (a dose of Co.No. 6-b that alone did not protect in the 6 Hz 44 mA model). Theco-administration of 10 mg/kg Co. No. 6-b produced an ED₅₀ in thelevetiracetam dose-response of 2.4 mg/kg (˜100-fold lower compared withlevetiracetam alone) and importantly yielded full protection in the 6 Hz44 mA seizure model. These results are suggestive of a positivepharmacodynamic interaction in the 6 Hz seizure model between Co. No.6-b and levetiracetam.

The results of the studies performed with compound 6-b are listed inTables 11-13 below.

TABLE 11 Time-to-Peak Effect Determination for Co. No. 6-b (p.o.) in the6 HZ (32 mA) Assay. Dose Time # protected/ # rotarod motor (mg/kg, p.o.)(h) # tested impairment/# tested 10 0.25 1/4 0/4 0.5 3/4 0/4 1 0/4 0/4 21/4 0/4 4 0/4 0/4 20 0.25 4/4 0/4 0.5 3/4 0/4 1 4/4 0/4 2 0/4 0/4 4 1/40/4 TPE determined to be 0.5 h.

TABLE 12 Dose-Response Study for Co. No. 6-b in the 6 Hz Assay (32 mAand 44 mA; 0.5 h TPE). Dose # protected/ # rotarod motor Test (mg/kg,p.o.) # tested impairment/# tested 6 Hz 20 7/8 0/8 32 mA 10 6/8 0/8 52/8 0/8 2.5 1/8 0/8 ED₅₀ (95% CI): 7.2 mg/kg (4.2 to 11.8) 6 Hz 40 8/80/8 44 mA 20 6/8 0/8 15 4/8 0/8 10 0/8 0/8 ED₅₀ (95% CI): 16.1 mg/kg(13.0 to 20.1)

TABLE 13 Combination Studies for Co. No. 6-b with LEV in the 6 Hz Assay(44 mA). Dose # protected/ # rotarod motor Drug (mg/kg) # testedimpairment/# tested LEV 200 2/8 0/8 400 4/9 0/9 800 10/12  0/12 ED₅₀(95% CI): 345.4 mg/kg (211.0 to 485.3) LEV + Co. No. 6-b 200 8/8 0/8 10mg/kg 100 8/8 0/8 50 5/8 0/8 10 5/8 0/8 1 5/8 0/8 ED₅₀ (95% CI): 2.4(0.0-6.4) Co. No. 6-b (p.o.) 10 mg/kg tested in combination with LEV(i.p.) Co. No. 6-b 10 mg/kg, not active when administered alone

Example 4—Studies with Compound LY404039

3.1. Combination Study with LY404039 and Levetiracetam

LY-404039 was tested alone and in combination with levetiracetamaccording to the procedures already described hereinabove. The resultsof the studies performed with LY-404039 are listed in tables 14-15.

TABLE 14 Dose-Response Studies for LY404039 in the 6 Hz Assay (32 mA and44 mA). A time-to-peak effect of 0.5 h was determined in the 32 mA 6 Hztest (s.c.) and used for 6 Hz (32 mA and 44 mA) studies. Dose #protected/ # rotarod motor Test (mg/kg, s.c.) # tested impairment/#tested 6 Hz 40 8/8 1/8 32 mA 20 6/8 1/8 10 5/8 0/8 5  1/16  1/16 ED₅₀(95% CI): 10.9 mg/kg (7.8 to 15.9) 6 Hz 40 7/8 2/8 44 mA 20 7/8 1/8 103/8 1/8 5  0/16  0/16 ED₅₀ (95% CI): 14.1 mg/kg (10.0 to 20.6) TD₅₀: >40mg/kg^(a) ^(a)40 mg/kg - 3 out of 16 total (32 mA and 44 mA combined)with impairment. Note: no activity observed following vehicleadministration in 32 or 44 mA. Dose selected for combination studieswith LEV in 6 Hz (44 mA): LY404039 5 mg/kg.

TABLE 15 Combination Studies for LY404039 with Levetiracetam (LEV) inthe 6 Hz (44 mA) Assay. Dose # protected/ # rotarod motor Drug (mg/kg) #tested impairment/# tested LEV^(a) 200 2/8 0/8 400 4/9 0/9 800 10/12 0/12 LEV ED₅₀ (95% CI): 345.4 mg/kg (211.0 to 485.3) LEV + 200 8/8 0/8LY404039 50 6/8 1/8 5 mg/kg^(b) 20 6/8 2/8 5 2/8 1/8 LEV ED₅₀ (95% CI):12.8 mg/kg (2.5-25.2) ^(a)LEV alone shown previously, confirmation dosesperformed in combination with Co. No. 2-a (see previous table above).^(b)LY404039 (s.c.) 5 mg/kg tested in combination with LEV (i.p.);LY404039 5 mg/kg was not active when administered alone. Additional LEV(low-dose) control groups were tested at 25 and 6.25 mg/kg (1/8 and 0/6protected, respectively). Vehicle-treated mice (10% sterile water -NaCl; s.c., 0.5 h TPE and 0.5% MC, i.p., 1 h TPE) showed no protectionor rotarod impairment.

At a dose of 5 mg/kg LY404039 increases the potency of LEV, leading toan approximate 27-fold shift in the ED₅₀. This suggests a positivepharmacodynamic relationship.

Example 5—Reference Studies with Compound CAS 1092453-15-0 5.1.Combination Study with2,3-dihydro-7-methyl-5-[3-(1-piperazinylmethyl)-1,2,4-oxadiazol-5-yl]-2-[[4(trifluoromethoxy)phenyl]methyl]-1H-isoindol-1-one [CAS 1092453-15-0](Described in WO 2008150233, WO 2011084098) and Levetiracetam

CAS 1092453-15-0 was tested alone and in combination with levetiracetamaccording to the procedures already described hereinabove. The resultsof example 5 are listed in tables 16-17.

TABLE 16 Dose-Response Studies for CAS 1092453- 15-0 in the 6 Hz Assay(32 mA). Dose Time # protected/ # rotarod motor (mg/kg, s.c.) (h) #tested impairment/# tested 20 0.25 1/4 0/4 0.5 0/4 0/4 1 1/4 0/4 2 0/40/4 4 0/4 0/4 40 0.25 1/4 0/4 0.5 1/4 0/4 1 1/4 0/4 2 0/4 0/4 4 0/4 0/480 0.25 0/4 0/4 0.5 0/4 0/4 1 1/4 1/4

Low activity was observed at doses and time points tested. Greatestactivity at 0.25-1 h in tested doses. Combination studies were performedusing 20 mg/kg, s.c, 1 h TPE in the 6 Hz (44 mA) assay.

TABLE 17 Combination Studies for CAS 1092453-15-0 with Levetiracetam(LEV) in the 6 Hz (44 mA) Assay. LEV Dose # protected/ # rotarod motorDrug (mg/kg) # tested impairment/# tested LEV^(a) 200 2/8 0/8 400 4/90/9 800 10/12  0/12 LEV ED₅₀ (95% CI): 345.4 mg/kg (211.0 to 485.3) [CAS1092453- 0/8 0/8 15-0] (20 mg/kg, alone) LEV + [CAS 400 4/8 0/81092453-15-0] 200 5/8 0/8 20 mg/kg^(b) 50 3/8 0/8 20 2/8 0/8 5 1/8 1/8LEV ED₅₀ (95% CI): 238.9 mg/kg (41.6 - above highest dose tested)^(a)Additional LEV (low-dose) control groups were tested at 25 and 6.25mg/kg (1/8 and 0/6 protected, respectively). ^(b)[CAS 1092453-15-0] 20mg/kg (s.c.; 1 h TPE) tested in combination with LEV (i.p.; 1 h TPE);[CAS 1092453-15-0] 20 mg/kg displayed low activity when administeredalone (6 Hz, 32 mA), and it was not tested in 6 Hz (44 mA). Thiscompound displayed an in vitro EC₅₀ = 562 nM (E_(max) = 197%) whentested in the GTPγS assay described hereinbefore and no occupancy wasobserved in ex vivo experiments in rats. Note: Vehicle-treated animals(10% HPβCD-NaCl, s.c., 1 h and 0.5% MC, i.p., 1 h) showed no protectionor motor impairment, N = 8.

The current data set indicates that mGlu2 PAM or agonist molecules haveanticonvulsant activity in the 6 Hz animal model. Tested mGlu2 PAMs withEC₅₀ potencies ≤150 nM (as determined in the [³⁵S]GTPγS assay),appropriate PK parameters and brain penetration, showed activity in boththe 32 and 44 mA 6 Hz paradigm. Furthermore, all the tested moleculesshowed synergistic effects with LEV. In contrast, molecule CAS1092453-15-0, which was only weakly active (EC₅₀ 562 nM) in vitro, didnot show activity in either of the 6 Hz tests, and also did not displaysynergy with LEV.

Importantly, the data indicate that, under conditions of comparable PKcharacteristics and appropriate brain penetration, the most potent mGlu2PAMs, based on in vitro EC₅₀ values, also appeared most potent in vivo,suggesting that in vitro and in vivo potency can be linked. Moreover,synergistic effects with LEV were consistently seen with mGlu2 PAM dosessimilar to the ED₅₀ obtained in the 32 mA model or at least 2-fold loweras the ED₅₀ determined in the 44 mA paradigm (i.e. a dose inactive inthe 44 mA test when the molecules were tested alone).

Also for LY404039, the mGlu2/3 agonist, activity in both 6 Hz tests wasseen and synergy was seen at a dose 3-fold lower than the ED₅₀determined in the 44 mA model, which was inactive when tested alone.

Based on the available preclinical data in the 6 Hz 44 mA model, itseems that combining a potent SV2A ligand and a potent mGlu2 PAM, leadsto a decrease in the median effective dose or ED₅₀ of the SV2A ligand,such as LEV, between 35 and 100-fold.

Thus, while not wishing to be bound by theory, it is suggested thatpositive allosteric modulator of metabotropic glutamatergic receptorsubtype 2 (mGluR2 PAM) compounds, in particular mGluR2 PAM compoundshaving an EC₅₀ potency of ≤150 nM (as determined in the [³⁵S]GTPγSassay), wherein EC₅₀ is the concentration producing half-maximal effectin a concentration-response curve obtained in the presence of EC₂₀ ofglutamate, and appropriate PK parameters and brain penetration, resultin a synergistic combination with an SV2A ligand, in particularlevetiracetam, at non-effective doses of one or both of compound (a) andcompound (b) of the combination of the invention.

Thus, in a further embodiment, the positive allosteric modulator ofmetabotropic glutamatergic receptor subtype 2 (mGluR2 PAM) compound ofthe combination of the invention as defined herein is selected from anmGluR2 PAM compound having an EC₅₀ potency of ≤150 nM (as determined inthe [³⁵S]GTPγS assay), wherein EC₅₀ is the concentration producinghalf-maximal effect in a concentration-response curve obtained in thepresence of EC₂₀ of glutamate.

Prophetic Examples

A) Dominant-Submissive Relations (DSR) in Rat In Vivo Assay

The DSR assay is divided into two models: Reduction of Dominant BehaviorModel (RDBM) of mania and Reduction of Submissive Behavior Model (RSBM)of depression. The RDBM, wherein the dominant animals are treated withtest compound, is predictive of the ability of the test compound totreat mania. The RSBM, wherein the submissive animals are treated withtest compound, is predictive of the ability of the test compound totreat depression.

Male Sprague Dawley rats (140 to 160 g) from Charles River LaboratoriesWilmington, Mass. are used in this assay. Shipments of rats are receivedat two-week intervals. Each shipment will go through five-dayquarantine, one-week acclimation period and one-week selection process,followed by five-weeks of drug or vehicle treatment to those pairsselected.

Rats will be housed four per cage. Access to food will be restricted toone hour per day after testing on Monday through Thursday. After testingon Friday, rats will have free access to food until being fasted againon Sunday. At no time will the rats be deprived of water. The fooddeprivation periods used will have little effect on weight gain as theaverage weight of rats will be about 300 g by the end of the study. Atthe conclusion of experiment rats will be sacrificed by decapitation,the trunk blood and brains will be collected for in vitro experimentsand drug concentration measurements.

The basic testing apparatus consisted of two chambers connected with atunnel only large enough to allow one rat to pass through at a time. Onthe floor, at the mid-point of the tunnel will be a container ofsweetened milk. This basic apparatus will be replicated, so that a totalof four pairs of rats can be video tracked simultaneously. The cameracan distinguish rats marked by different colors. Thus, the rats' headswill be colored for the purpose of video tracking, red in one cage andyellow in the other cage. Only one animal at a time can have comfortableaccess to the feeder, but both animals can drink milk during thefive-minute daily session. During the five-minute daily sessions, timespent in the feeder zone by each rat will be recorded by the videotracking software and saved into a text file.

The test will begin with a random assignment of rats into pairs. Eachmember of a pair will be placed in an opposite chamber of the testingapparatus. The time spent in the feeder zone by each animal will berecorded. During the first week (five days) of testing the animalshabituate to the new environment. Dominance will be assigned to theanimal with the highest score during the second week of testing if threecriteria are achieved. First, there must be a significant difference(two-tailed t-test, P<0.05) between the average daily drinking scores ofboth animals. Second, the dominant animal score must be at least 25%greater than the submissive animal's score. Finally, there must be no“reversals” during the pair selection week where the putative submissiverat out-scored its dominant partner on isolated occasions. Ideally therewill be minimal reversals during the acclimation week as well. Onlyanimal pairs that achieve these criteria will be continued in the study.

Significant differences between time spent on the feeder by dominant andsubmissive rats will be determined by ANOVA using GraphPad Prismsoftware (GraphPad Software, Inc. San Diego, Calif.) followed by atwo-tailed t-test (P<0.05). Comparisons will be made between treatmentgroups using normalized dominance level values in paired animals. Thedominance level is a value that measures social relation between pairedsubjects. Dominance level (DL)=FTD−FTS where FTD is the feeder time ofdominant rats and FTS is the feeder time of submissive rats. Thenormalization will be conducted according to the formula:Dominance Level(week n in %)=(Dominance Level(week n))/(DominanceLevel(week 2)

The statistical significance of the difference in dominance levelbetween the control group (pairs of rats where both dominant andsubmissive animals will be treated with vehicle) and the treatment group(submissive rats will be treated with drug and dominant rats withvehicle) will be determined by ANOVA, followed by a t-test. The activityonset time value at 50% of response (AOT-50) and the minimum and maximumresponse to drug will be calculated based on the reduction of thedominance level value using non-linear regression analysis (GraphPadSoftware, Inc., San Diego, Calif.). The normalized DL values will beused for this calculation, where DL values for treatment weeks will benormalized as a percent of the second week (pretreatment) value of thatpair according the above formula. In these settings the minimum of theresponse (DL) determines drug positive activity, corresponding toefficacy, since DL values will be reduced if the response to a drug ispositive. In the case of the negative response to a drug (worsening ofsymptoms) DL values will be increased. If the drug does not have suchactivity the maximum of the response will not exceed 100%. Any maximalDL value significantly higher than control value (about 100%) indicatesdrug negative activity.

Levetiracetam and mGluR2 PAM/agonist compound (e.g. compounds 2, 2-a,25-a, 6-b or LY-404039) will be evaluated in the rat RDBM according tothe procedure described in more detail below.

Groups of dominant rats will be treated p.o. QD with levetiracetam 10mg/kg and mGluR2 PAM/agonist compound at various concentrations fromapproximately 0.05 mg/kg (n≥3), at 0.5 mg/kg (n≥3), at 2.5 mg/kg (n≥3),at 5.0 mg/kg (n≥3) and at 50.0 mg/kg (n≥3). A vehicle control group ofdominant rats will be treated with 0.5% methylcellulose (n≥3) and asecond control group of dominant rats will be treated i.p. QD withsodium valproate at 30 mg/kg (n≥6 from 2 studies of n≥3 each).

All treatments will be administered approximately 1 hour prior totesting. All treatments will be started on Saturday after the secondtesting week (selection week). The levetiracetam and mGluR2 PAM/agonistcompound will be administered orally (p.o.).

When dominant animals are treated with levetiracetam 10 mg/kg and mGluR2PAM/agonist compound the difference between dominant and submissive ratswill be lost after the first or second week of treatment depending onthe dosage. Similarly, when dominant animals are treated with sodiumvalproate, the difference between dominant and submissive rats will alsobe lost after first week of treatment. The permissiveness of thedominant rats treated with levetiracetam and mGluR2 PAM/agonist compoundor sodium valproate may be observed to increase. Thus the treateddominant rats will permit their submissive partners to increase theirtime on the feeder.

To compare different drug and dose effects the data will be normalizedto the initial control week values. The strongest effect oflevetiracetam and mGluR2 PAM/agonist compound combination will beobserved where there is a significant difference in dominance level (DL)values between vehicle and combination treated rats starting in thesecond week and continuing through the treatment duration of 5 weeks. Incomparison, animals (30 mg/kg) that will be treated with sodiumvalproate will consistently show a decreased dominance level after thesecond week of treatment with the effect increasing in the followingweeks.

To estimate activity onset time (AOT), daily average values for feedertime of dominant and submissive animal pairs will be plotted andsignificant differences between these two groups will be calculatedusing the two-tail t-test.

To compare activity onset time (AOT) between different treatments theactivity onset time will be estimated from the non-linear regressionfit. The non-linear regression model will fit for each drug, combinationand dose normalized daily DL values.

Effects of levetiracetam and mGluR2 PAM/agonist compound in the RDBM areexpected to be dose dependent.

In this assay, the combination of levetiracetam and mGlu2 PAM/agonistcompounds is expected to reduce dominant behavior indicating that thecombination is active as an anti-manic.

B) Oral Tablets

As a specific example of an oral composition, 100 mg of a mGluR2 15PAM/agonist compound is formulated with sufficiently finely dividedlactose to provide a total amount of 580 to 590 mg to fill a size O hardgel capsule.

While the foregoing specification teaches the principles of the presentinvention, with examples provided for the purpose of illustration, itwill be understood that the practice of the invention encompasses all ofthe usual variations, adaptations and/or modifications as come withinthe scope of the following claims and their equivalents.

The invention claimed is:
 1. A method for the treatment of epilepsycomprising administering to a patient in need thereof a therapeuticallyeffective amount of a combination comprising: (a) a synaptic vesicleprotein 2A (“SV2A”) ligand selected from the group consisting oflevetiracetam, brivaracetam; and (b) a positive allosteric modulator(“PAM”) of metabotropic glutamatergic receptor subtype 2 (“mGluR2”)compound of

or a pharmaceutically acceptable salt thereof.
 2. The method of claim 1wherein the SV2A ligand is levetiracetam.
 3. The method of claim 1wherein the SV2A ligand is brivaracetam.
 4. The method of claim 1wherein the combination of the SV2A ligand and the PAM of a mGluR2compound is administered simultaneously to treat epilepsy.
 5. The methodof claim 1 wherein the combination of the SV2A ligand and the PAM of amGluR2 compound is administered separately to treat epilepsy.
 6. Themethod of claim 1 wherein the combination of the SV2A ligand and the PAMof a mGluR2 compound is administered sequentially to treat epilepsy. 7.The method of claim 1 wherein the epilepsy is epilepsy with partialonset seizures.
 8. The method of claim 1 wherein the epilepsy isepilepsy with myoclonic seizures.
 9. The method of claim 1 wherein theepilepsy is epilepsy with primary generalized tonic-clonic seizures. 10.The method of claim 1 wherein the epilepsy is treatment resistantepilepsy.
 11. A method for the treatment of epilepsy comprisingadministering to a patient in need thereof a therapeutically effectiveamount of a combination comprising: (a) a SV2A ligand selected from thegroup consisting of levetiracetam, brivaracetam; and (b) a PAM of mGluR2compound of

or a pharmaceutically acceptable salt thereof.
 12. The method of claim11 wherein the SV2A ligand is levetiracetam.
 13. The method of claim 11wherein the SV2A ligand is brivaracetam.
 14. The method of claim 11wherein the combination of the SV2A ligand and the PAM of a mGluR2compound is administered simultaneously to treat epilepsy and relateddisorders.
 15. The method of claim 11 wherein the combination of theSV2A ligand and the PAM of a mGluR2 compound is administered separatelyto treat epilepsy and related disorders.
 16. The method of claim 11wherein the combination of the SV2A ligand and the PAM of a mGluR2compound is administered sequentially to treat epilepsy and relateddisorders.
 17. The method of claim 11 wherein the epilepsy is epilepsywith partial onset seizures.
 18. The method of claim 11 wherein theepilepsy is epilepsy with myoclonic seizures.
 19. The method of claim 11wherein the epilepsy is epilepsy with primary generalized tonic-clonicseizures.
 20. The method of claim 11 wherein the epilepsy is treatmentresistant epilepsy.